Site-specific incorporation of phosphoserine into proteins in escherichia coli

ABSTRACT

Nucleic acids encoding mutant elongation factor proteins (EF-Sep), phosphoseryl-tRNA synthetase (SepRS), and phosphoseryl-tRNA (tRNASep) and methods of use in site specific incorporation of phosphoserine into &amp; protein or polypeptide are described. Typically, SepRS preferentially aminoacylates tRNASepwith O-phosphoserine and the tRNASep recognizes at least one codon such as a stop codon. Due to the negative charge of the phosphoserine, Sep-tRNASep does not bind elongation factor Tu (EF-Tu). However, mutant EF-Sep proteins are disclosed that bind Sep-tRNASep and protect Sep-tRNASep from deacylation. In a preferred embodiment the nucleic acids are on vectors and are expressed in cells such as bacterial cells, archeaebacterial cells, and eukaryotic cells. Proteins or polypeptides containing phosphoserine produced by the methods described herein can be used for a variety of applications such as research, antibody production, protein array manufacture and development of cell-based screens for new drug discovery.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.61/390,853, filed on Oct. 7, 2010, and U.S. Provisional Application No.61/470,332, filed on Mar. 31, 2011.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government Support under Agreement R01GM022854 awarded by the National Institutes of Health and Agreement0654283 awarded by the National Science Foundation. The Government hascertain rights in the invention.

FIELD OF THE INVENTION

The field of the present invention generally relates to methods for thesite specific phosphorylation of proteins in vitro and in vivo.

BACKGROUND OF THE INVENTION

Signal transduction is any process by which a cell converts one kind ofsignal or stimulus into another. Processes referred to as signaltransduction often involve a sequence of biochemical reactions insidethe cell, which are carried out by enzymes and linked through secondmessengers. Signal transduction is often accomplished by the activationof enzymes that can act upon other enzymes and change their catalyticactivity. This may lead to increases or decreases in the activity ofcertain metabolic pathways, or may lead to even large intracellularchanges, for example, the initiation of specific patterns of geneexpression and/or changes in cell proliferation.

The most common covalent modification used in signal transductionprocesses is phosphorylation, which results in the alteration of theactivity of those enzymes which become phosphorylated. Phosphorylationis the addition of a phosphate (PO₄) group to a protein or a smallmolecule. Any of several amino acids in a protein may be phosphorylated.Phosphorylation on serine is the most common, followed by threonine.Tyrosine phosphorylation is relatively rare. However, since tyrosinephosphorylated proteins are relatively easy to purify using antibodies,tyrosine phosphorylation sites are relatively well understood. Histidineand aspartate phosphorylation occurs in prokaryotes as part oftwo-component signaling. Other types of phosphorylation includeoxidative phosphorylation. Adenosine triphosphate (ATP), the“high-energy” exchange medium in the cell, is synthesized in themitochondrion by addition of a third phosphate group to Adenosinediphosphate (ADP) in a process referred to as oxidative phosphorylation.ATP is also synthesized by substrate level phosphorylation duringglycolysis. ATP is synthesized at the expense of solar energy byphotophosphorylation in the chloroplasts of plant cells.

In eukaryotes, protein phosphorylation is probably the most importantregulatory event. Many enzymes and receptors are switched “on” or “off”by phosphorylation and dephosphorylation. Phosphorylation is catalyzedby enzymes known as ATP-dependent phosphotransferases which are oftensimply referred to as “kinases.” These include, among others, proteinkinases, lipid kinases, inositol kinases, non-classical protein kinases,histidine kinases, aspartyl kinases, nucleoside kinases, andpolynucleotide kinases.

Phosphorylation regulates protein function, for example, by affectingconformation. This in turn regulates such processes as enzyme activity,protein-protein interactions, subcellular distribution, and stabilityand degradation. The stoichiometry of phosphorylation of a given site iscontrolled by the relative activities of a cell's repertoire of proteinkinases and phosphatases. Thus phosphorylation can often generateextremely rapid and reversible changes in the activity of targetproteins. The ability to assay the state of phosphorylation of specificproteins is of great utility in the quest to establish the function of agiven protein. Such assays are also critical for the identification ofdrugs that can influence the phosphorylation, and hence the function, ofspecific proteins.

In general, phosphoproteins are highly unstable and difficult toproduce, both in terms of specific phosphorylation of biologicallyrelevant amino acids and subsequent purification of protein. A means tospecify and drive a targeted phosphorylation event with a high degree ofcertainty and efficiency is needed. This is particularly important forrecombinant proteins expressed in bacterial or fungal expression systemswhich do not phosphorylate proteins in the same way as mammalian cells.

Therefore, it is an object of the present invention to provide a methodfor the site specific phosphorylation of proteins.

It is further an object of the present invention to provide a method forthe site specific phosphorylation of proteins in vivo.

In particular, it is an object of the present invention to provide amethod for the site specific incorporation of phosphoserine into aprotein.

SUMMARY OF THE INVENTION

Mutant elongation factor proteins (EF-Sep) are described for use withphosphoseryl-tRNA synthetase (SepRS) and phosphoseryl-tRNA (tRNA^(Sep))in site specific incorporation of phosphoserine into a protein orpolypeptide. Typically. SepRS preferentially aminoacylates tRNA^(Sep)with O-phosphoserine and the tRNA^(Sep) recognizes at least one codonsuch as a stop codon. Due to the negative charge of the phosphoserine,Sep-tRNA^(Sep) does not bind elongation factor Tu (EF-Tu). However, thedisclosed EF-Sep proteins can bind Sep-tRNA^(Sep) and protectSep-tRNA^(Sep) from deacylation and catalyze the covalent transfer ofthe phosphoserine amino acid onto the polypeptide.

In some embodiments, EF-Sep is a mutant form of bacterial EF-Tu having amutation at one or more of amino acid residues corresponding to His67,Asp216, Glu217, Phe219, Thr229, and Asn274 in E. coli EF-Tu, which arelocated in the amino acid binding pocket for aminoacylated tRNA. In someembodiments, EF-Sep is a mutant form of eukaryotic elongation factor 1A(eEF1A) with mutations in positions equivalent to bacterial counterpart.In preferred embodiments, the EF-Sep has the amino acid sequence SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or a conservative variantthereof. Nucleic acids encoding EF-Sep are also disclosed. For example,in some embodiments, the nucleic acid sequence encoding EF-Sep has thenucleic acid sequence SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, or a conservative variant thereof.

In a preferred embodiment, “tRNA^(Sep)” and “SepRS” refer to thecysteinyl-tRNA from Methanocaldococcus jannaschii and thephosphoseryl-tRNA synthetase from Methanococcus maripaludis,respectively and variants thereof having conservative substitutions,additions, and/or deletions therein not affecting the structure orfunction. Typically, SepRS preferentially aminoacylates tRNA^(Sep) withO-phosphoserine and the tRNA^(Sep) recognizes at least one codon. In apreferred embodiment, the tRNA^(Sep) recognizes a stop codon or anunconventional or non-native codon.

Methods for producing target proteins that contain at least onephosphoserine are described. The method results in proteins that have aphosphoserine incorporated into a protein in a manner indistinguishablefront the phosphorylation of a serine by a kinase. Nucleic acidsencoding genes with SepRS and tRNA^(Sep) activity are provided,preferably on vectors, such as cloning vectors and expression vectors.These vectors can be in the form of a plasmid, a bacterium, a virus, anaked polynucleotide, or a conjugated polynucleotide. In one embodiment,the vectors are expressed in cells such as bacterial cells (e.g.,Escherichia coli), archeaebacterial cells, and eukaryotic cells (e.g.,yeast cells, mammalian cells, plant cells, insect cells, fungal cells).The cells preferably lack a protein with Sep-tRNA:Cys-tRNA synthase(SepCysS) activity that converts tRNA-bound phosphoserine to cysteine.In an alternative embodiment, the vectors are expressed in an in vitrotranscription/translation system. In this embodiment the vectors aretranscribed and translated prior to or along with nucleic acids encodingone or more proteins or polypeptides.

In some embodiments, the target protein containing phosphoserine isproduced and modified in a cell-dependent manner. This provides for theproduction of proteins that are stably folded, glycosylated, orotherwise modified by the cell.

Kits for producing polypeptides and/or proteins containing phosphoserineare also provided.

The proteins or polypeptides containing phosphoserine and antibodies tosuch polypeptides or proteins have a variety of uses including the studyof kinases, phosphotases, and target proteins in signal transductionpathways, antibody production, protein array manufacture and developmentof cell-based screens for new drug discovery and the development oftherapeutic agents, agricultural products, or peptide-based librariessuch as phage display libraries.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a diagram showing the indirect pathway for the synthesis ofCys-tRNA^(Cys) in methanogenic archaea. FIG. 1B is a diagram showing thesecondary structure (of Methanocaldococcus jannaschii tRNA^(Cys) (SEQ IDNO:41) shown in clover leaf form. Mutations introduced to formtRNA^(Sep) are indicated with arrows. FIG. 1C is a graph showing percentphosphoserine (Sep) acceptance in M. jannaschii tRNA as a function oftime (min) for unfractionated total tRNA from E. coli (triangle) or E.coli strains expressing tRNA^(Cys) (closed circle) or tRNA^(Sep) (opencircle).

FIG. 2 is a graph showing chloramphenicol resistance (IC50, μg-ml) forE. coli. containing 1) a chloramphenicol acetyltransferase (CAT) genewith an amber stop code (UAG) at a permissive site and 2) combinationsof tRNA^(Sep), [SepRS or CysRS (Mmp)], SepCysS, and [EF-Sep or and EF-Tu(wt)]. The suppressor tRNA^(Sep) was coexpressed with the indicatedenzymes in E. coli Top 10ΔserB. Selection was carried out on LB agarplates containing 2 mM Sep and various concentrations ofchloramphenicol.

FIG. 3 is a graph showing deacylation of [¹⁴C]Sep-tRNA^(Cys) (percentSep-tRNA^(Cys) remaining) as a function of time following incubated inthe presence and absence of bovine serum albumin control (open circle),wild type EF-Tu (closed circle), or EF-Sep (square).

FIG. 4 is a graph showing kinase activity (phosphate incorporation intoMyBP (pmol/min)) as a function of MEK1 concentration (μg/assay) for wildtype (triangle) and mutant (closed and open circles) MEK1. Human MEK1was produced as a maltose-binding protein (MBP) fusion-protein in E.coli. Residues Ser218 and Ser222, which are targets of phosphorylationby MEK1 activators were either mutated to Glu218/Glu222 (closed circle)or to Sep218/Glu222 (open circle) to produce active MEK1 variants.Various amounts of MBP-MEK1 were used to phosphorylate inactive ERK2 invitro. ERK2 activity was then measured in a radiometric assay using[³²P]-γATP and myelin basic protein as substrates.

FIGS. 5A and 5B are graphs showing EF-Tu protects Cys-tRNA^(Cys) (FIG.5A) but not Sep-tRNA^(Cys) (FIG. 5B) from deacylation. Hydrolysis of M.jannaschii [³⁵S]Cys-tRNA^(Cys) or [¹⁴C]Sep-tRNA^(Cys) was determined atpH 8.2 and room temperature in the presence or absence of EF-Tu.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The term “transfer RNA (tRNA)” refers to a set of genetically encodedRNAs that act during protein synthesis as adaptor molecules, matchingindividual amino acids to their corresponding codon on a messenger RNA(mRNA). In higher eukaryotes such as mammals, there is at least one tRNAfor each of the 20 naturally occurring amino acids. The 3′ end of a tRNAis aminoacylated by a tRNA synthetase so that an amino acid is attachedto the 3′ end of the tRNA. This amino acid is delivered to a growingpolypeptide chain as the anticodon sequence of the tRNA reads a codontriplet in an mRNA.

The term “aminoacyl tRNA synthetase (AARS)” refers to an enzyme thatcatalyzes the esterification of a specific amino acid or its precursorto one of all its compatible cognate tRNAs to form an aminoacyl-tRNA.These charged aminoacyl tRNAs then participate in mRNA translation andprotein synthesis. The AARS show high specificity for charging aspecific tRNA with the appropriate amino acid. In general, there is atleast one AARS for each of the twenty amino acids.

The term “tRNA^(Sep)” refers to a tRNA that can be aminoacylated withO-phosphoserine (Sep) and recognize at least one codon such that thephosphoserine is incorporated into a protein or polypeptide. In someembodiments, the tRNA^(Sep) is a tRNA^(Cys) from Methanocaldococcusjannaschii containing a C20U mutation that improves aminoacylation bySepRS without affecting CysRS recognition. In some embodiments, thetRNA^(Sep) contains an anticodon that binds a stop codon.

The term “Sep-tRNA^(Sep)” refers to a tRNA^(Sep) that has beenaminoacylated with O-phosphoserine (Sep).

The term “O-phosphoseryl-tRNA synthetase (SepRS)” refers to anO-phosphoseryl-tRNA synthetase that preferentially aminoacylatestRNA^(Sep) with O-phosphoserine (Sep) to form Sep-tRNA.

The term “EF-Sep” refers to a mutant elongation factor protein thatbinds Sep-tRNA^(Sep) and catalyzes the covalent transfer of thephosphoserine amino acid onto the polypeptide. Due to the negativecharge of the phosphoserine, Sep-tRNA^(Sep) does not bind elongationfactor Tu (EF-Tu). EF-Sep proteins can bind Sep-tRNA^(Sep), protectSep-tRNA^(Sep) from deacylation, and catalyze the covalent transfer ofthe phosphoserine amino acid onto the polypeptide.

As used herein “suppressor tRNA” refers to a tRNA that alters thereading of a messenger RNA (mRNA) in a given translation system. Forexample, a suppressor tRNA can read through a stop codon.

The term “anticodon” refers to a unit made up of three nucleotides thatcorrespond to the three bases of a codon on the mRNA. Each tRNA containsa specific anticodon triplet sequence that can base-pair to one or morecodons for an amino acid or “stop codon.” Known stop codons include butare not limited to, the three codon bases UAA (known as ochre), UAG(known as amber), and UGA (known as opal), which do not code for anamino acid but act as signals for the termination of protein synthesis.

The term “protein” “polypeptide” or “peptide” refers to a natural orsynthetic molecule comprising two or more amino acids linked by thecarboxyl group of one amino acid to the alpha amino group of another.

The term “residue” as used herein refers to an amino acid that isincorporated into a protein. The amino acid may be a naturally occurringamino acid and, unless otherwise limited, may encompass known analogs ofnatural amino acids that can function in a similar manner as naturallyoccurring amino, acids.

The term “polynucleotide” or “nucleic acid sequence” refers to a naturalor synthetic molecule comprising two or more nucleotides linked by aphosphate group at the 3′ position of one nucleotide to the 5′ end ofanother nucleotide. The polynucleotide is not limited by length, andthus the polynucleotide can include deoxyribonucleic acid (DNA) orribonucleic acid (RNA).

The term “gene” refers to a polynucleotide that encodes a protein orfunctional RNA molecule.

The term “vector” or “construct” refers to a polynucleotide capable oftransporting into a cell another polynucleotide to which the vectorsequence has been linked. The term “expression vector” includes anyvector, (e.g., a plasmid, cosmid or phage chromosome) containing a geneconstruct in a form suitable for expression by a cell (e.g., linked to atranscriptional control element). “Plasmid” and “vector” are usedinterchangeably, as a plasmid is a commonly used form of vector.

The term “operatively linked to” refers to the functional relationshipof a nucleic acid with another nucleic acid sequence. Promoters,enhancers, transcriptional and translational stop sites, and othersignal sequences are examples of nucleic acid sequences operativelylinked to other sequences. For example, operative linkage of gene to atranscriptional control element refers to the physical and functionalrelationship between the gene and promoter such that the transcriptionof the gene is initiated front the promoter by an RNA polymerase thatspecifically recognizes, binds to and transcribes the DNA.

The terms “transformation” and “transfection” refer to the introductionof a polynucleotide, e.g., an expression vector, into a recipient cellincluding introduction of a polynucleotide to the chromosomal DNA of thecell.

The term “variant” refers to an amino acid or nucleic acid sequencehaving conservative substitutions, non-conservative subsitutions (i.e. adegenerate variant), substitutions within the wobble position of a codonencoding an amino acid, amino acids added to the C-terminus of apeptide, or a peptide having 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or99% sequence identity to an amino acid sequence.

The term “conservative variant” refers to a particular nucleic acidsequence that encodes identical or essentially identical amino acidsequences. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. The following sets forthexemplary groups which contain natural amino acids that are“conservative substitutions” for one another. Conservative SubstitutionGroups 1 Alanine (A) Serine (S) Threonine (T); 2 Aspartic acid (D)Glutamic acid (E); 3 Asparagine (N) Glutamine (Q); 4 Arginine (R) Lysine(K); 5 Isoleucine (I) Leucine (L) Methionine (M) Valine (V); and 6Phenylalanine (F) Tyrosine (Y) Tryptophan (W).

The term “percent (%) sequence identity” or “homology” refers to thepercentage of nucleotides or amino acids in a candidate sequence thatare identical with the nucleotides or amino acids in a reference nucleicacid sequence, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. Alignmentfor purposes of determining percent sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN,ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters formeasuring alignment, including any algorithms needed to achieve maximalalignment over the full-length of the sequences being compared can bedetermined by known methods.

The term “translation system” refers to the components necessary toincorporate an amino acid into a growing polypeptide chain (protein).Components of a translation system generally include amino acids,ribosomes, tRNAs, synthetases, and mRNA. The components described hereincan be added to a translation system, in vivo or in vitro, toincorporate phosphoserine into a protein.

The term “transgenic organism” refers to any organism, in which one ormore of the cells of the organism contains heterologous nucleic acidintroduced by way of human intervention, such as by transgenictechniques well known in the art. The nucleic acid is introduced intothe cell, directly or indirectly by introduction into a precursor of thecell, by way of deliberate genetic manipulation, such as bymicroinjection or by infection with a recombinant virus. Suitabletransgenic organisms include, but are not limited to, bacteria,cyanobacteria, fungi, plants and animals. The nucleic acids describedherein can be introduced into the host by methods known in the art, forexample infection, transfection, transformation or transconjugation.

The term “eukaryote” or “eukaryotic” refers to organisms or cells ortissues derived from these organisms belonging to the phylogeneticdomain Eukarya such as animals (e.g., mammals, insects, reptiles, andbirds), ciliates, plants (e.g., monocots, dicots, and algae), fungi,yeasts, flagellates, microsporidia, and protists.

The term “prokaryote” or “prokaryotic” refers to organisms including,but not limited to, organisms of the Eubacteria phylogenetic domain,such as Escherichia coli, Thermus thermophilus, and Bacillusstearothermophilus, or organisms of the Archaea phylogenetic domain suchas, Methanococcus jannaschii, Methanobacterium thermoautotrophicum,Halobacterium such as Haloferax volcanii and Halobaeterium speciesNRC-1. Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcushorikoshii, and Aeuropyrum pernix.

II. Compositions A. Aminoacyl-tRNA Synthetases

A tRNA that can be aminoacylated with O-phosphoserine (“tRNA^(Sep)” ) isdisclosed for use in incorporating phosphoserine into a protein. ThetRNA^(Sep) recognizes at least one codon in the mRNA for the proteinsuch that a phosphoserine can incorporated into the protein. Forexample, the tRNA^(Sep) can contain an anticodon that binds a stop codonor an unconventional or non-native codon, in some embodiments, thetRNA^(Sep) is a tRNA^(Cys) from an achaea, such as Methanocaldococcusjannaschii or Methanococcus maripaludis. tRNA^(Cys) is also found inMethanopyrus kandleri, Methanococcoides burtonii, Methanospirillumhungatei, Methanocorpusculum labreanum, Methanoregula boonei,Methanococcus aeolicus, Methanococcus vannieli, Methanosarcina mazei.Methanosarcina barkeri. Methanosarcina acetivorans, Methanosaetathermophila, Methanoculleus marisnigri, Methanocaldococcus vulcanius,Methanocaldococcus fervens, and Methanosphaerula palustris. In preferredembodiments, the tRNA^(Sep) contains a mutation (e.g., C20U mutation)that improves aminoacylation by SepRS without affecting CysRSrecognition. In particularly preferred embodiments, the tRNA^(Sep) isencoded by the nucleic acid sequence SEQ ID NO:41, or a conservativevariant thereof.

tRNA^(Sep) from Methanocaldococcus jannaschii (FIG.1B):_________________

(SEQ ID NO: 41) GCCGGGGTAGTCTAGGGGTTAGGCAGCGGACTGCAGATCCGCCTTACGTGGGTTCAAATCCCACCCCCGGCT

A phosphoseryl-tRNA synthetase (SepRS) that preferentially aminoacylatestRNA^(Sep) with phosphoserine is also disclosed for use in incorporatingphosphoserine into a protein. In some embodiments, the SepRS is aphosphoseryl-tRNA synthetase from an achaea, such as Methanococcusmaripaludis or Methanocaldococcus jannaschii. SepRS is also found inMethanopyrus kandleri, Methanococcoides burtonii, Methanospirillumhungatei, Methanocorpusculum labreanum, Methanoregula boonei,Methanococcus aeolicus, Methanococcus vannieli, Methanosarcina mazei,Methanosarcina barkeri, Methanosarcina acetivorans, Methanosaetathermophila, Methanoculleus marisnigri, Methanocaldococcus vulcanius,Methanocaldococcus fervens, and Methanosphaerula palustris.

In particularly preferred embodiments, the SepRS has the amino acidsequence SEQ ID NO:43 or 46, or a conservative variant thereof. Forexample, the SepRS can be encoded by the nucleic acid sequence SEQ IDNO:42 or 45, or a variant thereof.

SepRS from Methanocaldococcus jannaschii: (SEQ ID NO: 42)ATGAAATTAAAACATAAAAGGGATGATAAAATGAGATTTGATATAAAAAAGGTTTTAGAGTTAGCAGAGAAGGATTTTGAGACGGCATGGAGAGAGACAAGGGCATTAATAAAGGATAAACATATTGACAATAAATATCCAAGATTAAAGCCTGTCTATGGAAAGCCACATCCAGTGATGGAGACGATAGAGAGATTAAGACAAGCTTATCTAAGAATGGGATTTGAAGAGATGATTAATCCAGTTATCGTTGATGAGATGGAGATTTATAAGCAATTTGGACCAGAAGCAATGGCAGTTTTAGATAGATGTTTTTACTTGGCTGGATTACCAAGGCCAGATGTTGGTTTAGGAAATGAGAAGGTTGAGATTATAAAAAATTTGGGCATAGATATAGATGAGGAGAAAAAAGAGAGGTTGAGAAGTTTTACATTTATACAAAAAAGGAGCTATAGATGGGGATGATTTAGTCTTTGQAGATTGCCAAAGCTTTAAATGTGAGTAATGAAATGGGATTGAAGGTTTTAGAAACTGCATTTCCTGAATTTAAAGATTTGAAGCCAGAATCAACAACTCTAACTTTAAGAAGCCACATGACATCTGGGTGGTTTATAACTCTAAGCAGTTTAATAAAGAAGAGAAAACTGCTTTAAAGTTATTCTCTATAGATAGATGTTTTAGAAGGGAGCAAAGAGAGGATAGAAGCCATTTAATGAGTTATCACTCTGCATCTTGTGTAGTTGTTGGTGAAGATGTTAGTGTAGATGATGGAAAGGTAGTTGCTGAAGGATTGTTGGCTCAATTTGGATTTACAAAATTTAAGTTTAAGCCAGATGAGAAAAAGAGTAAGTATTATACACCAGAAACTCAAACAGAGGTTTATGCCTATCATCCAAAGTTGGGAGAGTGGATTGAAGTAGCAACCTTTGGAGTTTATTCACCAATTGCATTAGCTAAATATAACATAGATGTGCCAGTTATGAACCTTGGCCTTAGGAGTTGAGAGGTTGGCAATGATTATTTACGGCTATGAGGATGTTAGGGCAATGGTTTATCCTCAATTTTATGAATACAGGTTGAGTGATAGAGATATAGCTGGGATGATAAGAGTTGATAAAGTTCCTATATTGGATGAATTCTACAACTTTGCAAATGAGCTTATTGATATATGCATAGCAAATAAAGATAAGGAAAGCCCATGTTCAGTTGAAGTTAAAAGGGAATTCAATTTCAATGGGGAGAGAAGAGTAATTAAAGTAGAAATATTTGAGAATGAACCAAATAAAAAGCTTTTAGGTCCTTCTGTGTTAAATGAGGTTTATGTCTATGATGGAAATATATATGGCATTCCGCCAACGTTTGAAGGGGTTAAAGAACAGTATATCCCAATTTTAAAGAAAGCTAAGGAAGAAGGAGTTTCTACAAACATTAGATACATAGATGGGATTATCTATAAATTAGTAGCTAAGATTGAAGAGGCTTTAGTTTCAAATGTGGATGAATTTAAGTTCAGAGTCCCAATAGTTAGAAGTTTGAGTGACATAAACCTAAAAATTGATGAATTGGCTTTAAAACAGATAATGGGGGAGAATAAGGTTATAGATGTTAGGGGACCAGTTTTCTTAAATGCAAAGGTTGAGATAAAATAG; (SEQ ID NO: 43)MKLKLHRDDKMRFDIKKVLELAEKDFETAWRETRALIKDKHIDNKYPRLKPVYGKPHPVMETIERLRQAYLRMGFEEMINPVIVDEMEIYKQFGPEAMAVLDRVFYLAGLPRDVGLGNEKVEIIKNLGIDIDEEKKERLREVLHLYKKGAIDGDDLVFEIAKALNVSNEMGLKVLETAFPEFKDLKPESTTLTLRSHMTSGWFTTLSSLIKKRKLPLKLFSIDRCFRREQREDRSHLMSYHSASCVVVGEDVSVDDGKVVAEGLLAQFGFTKFKFKPDEKKSKYYTPETQTEVYAYHPKLGEWIEVATFGVYSPIALAKYNIDVPVMNLGLGVERLAMIIYGYEDVRAMVYPQFYEYRLSDRDIAGMIRVDKVPILDEFYNFANELIDICIANKDKESPCSVEVKREFNFNGERRVIKVEIFENEPNKKLLGPSVLNEVYVYDGNIYGIPPTFEGVKEQYIPILKKAKEEGVSTNIRYIDGIIYKLVAKIEEALVSNVDEFKFRVPIVRSLSDINLKIDELALKQIMGENKVIDVRGPVFLNAKVEIK.SepRS from Methanococcus maripaludis: (SEQ ID NO: 45)ATGTTTAAAAGAGAAGAAATCATTGAAATGGCCAATAAGGACTTTGAAAAAGCATGGATCGAAACTAAAGACCTTATAAAAGCTAAAAAGATAAACGAAAGTTACCCAAGAATAAAACCAGTTTTGGAAAAACACACCCTGTAAATGACACTATTGAAAATTTAAGACAGGCATATCTTAGAATGGGTTTTGAAGAATATAAACCCAGTAATTGTCGATGAAAGAGATATTTATAAACAATTCGGCCCAGAAGCTATGGCAGTTTTGGATAGATGCTTTTATTTAGCGGGACTTCCAAGACCTGACGTTGGTTTGAGCGATGAAAAAATTTCACAGATTGAAAAACTTGGAATTAAAGTTTCTGAGCACAAAGAAAGTTTACAAAAAATACTTCACGGATACAAAAAAGGAACTCTTGATGGTGACGATTTAGTTTTAGAAATTTCAAATGCACTTGAAATTTCAAGCGAGATGGGTTTAAAAATTTTAGAAGATGTTTTCCCAGAATTTAAGGATTTAACCGCAGTTTCTTCAAAATTAACTTTAAGAAGCCACATGACTTCAGGATGGTTCCTTACTGTTTCAGACCTCATGAACAAAAAACCCTTGCCATTTAAACTCTTTTCAATCGATAGATGTTTTAGAAGAGAACAAAAAGAAGATAAAAGCCACTTAATGACATACCACTCTGCATCCTGTGCAATTGCAGGTGAAGGCGTGGATATTAATGATGGAAAAGCAATTGCAGAAGGATTATTATCCCAATTTGGCTTTACAAACTTTAAATTCATTCCTGATGAAAAGAAAAGTAAATACTACACCCCTGAAACACAGACTGAAGTTTACGCATACCACCCAAAATTAAAAGAATGGCTCGAAGTTGCTACATTTGGAGTATATTCGCCAGTTGCATTAAGCAAATACGGAATAGATGTACCTGTAATGAATTTGGGTCTTGGTGTTGAAAGACTTGCAATGATTTCTGGAAATTTCGCAGATGTTCGAGAAATGGTATATCCTCAGTTTTACGAACACAAACTTAATGACCGGAATGTCGCTTCAATGGTAAAACTCGATAAAGTTCCAGTAATGGATGAAATTTACGATTTAACAAAAGAATTAATTGAGTCATGTGTTAAAAACAAAGATTTAAAATCCCCTTGTGAATTAGCTATTGAAAAAACGTTTTCATTTGGAAAAACCAAGAAAAATGTAAAAATAAACATTTTTGAAAAAGAAGAAGGTAAAAATTTACTCGGACCTTCAATTTTAAACGAAATCTACGTTTACGATGGAAATGTAATTGGAATTCCTGAAAGCTTTGACGGAGTAAAAGAAGAATTTAAAGACTTCTTAGAAAAAGGAAAATCAGAAGGGGTAGCAACAGGCATTCGATATATCGATGCGCTTTGCTTTAAAATTACTTCAAAATTAGAAGAAGCATTTGTGTCAAACACTACTGAATTCAAAGTTAAAGTTCCAATTGTCAGAAGTTTAAGCGACATTAACTTAAAAATCGATGATATCGCATTAAAACAGATCATGAGCAAAAATAAAGTAATCGACGTTAGAGGCCCAGTCTTTTAAATGTCGAAGTAAA AATTGAATAA;(SEQ ID NO: 46) MFKREEIIEMANKDFEKAWIETKDLIKAKKINESYPRIKPVFGKTHPVNDTIENLRQAYLRMGFEEYINPVIVDERDIYKQFGPEAMAVLDRCFYLAGLPRPDVGLSDEKISQIEKLGIKVSEHKESLQKILHGYKKGTLDGDDLVLEISNALEISSEMGLKILEDVFPEFKDLTAVSSKLTLRSHMTSGWFLTVSDLMNKKPLPFKLFSIDRCFRREQKEDKSHLMTYHSASCAIAGEGVDINGDKAIAEGLLSQFGFTNFKFIPDEKKSKYYTPETQTEVYAYHPKLKEWLEVATFGVYSPVALSKYGIDVPVMNLGLGVERLAMISGNFADVREMVYPQFYEHKLNDRNVASMVKLDKVPVMDEIYDLTKELIESCVKNKDLKSPCELAIEKTFSFGKTKKVKINIFEKEEGKNLLGPSILNEIYVYDGNVIGIPESFDGVKEEFKDFLEKGKSEGVATGIRYIDALVGKITSKLEEAFVSNTTEFKVKVPIVRSLSDINLKIDDIALKQIMSKNKVIDVRGPVFLNVEVKIE.

B. Elongation Factor Proteins

Nucleic acid sequences encoding mutant elongation factor proteins(EF-Sep) are described for use with phosphoseryl-tRNA synthetase (SepRS)and phosphoseryl-tRNA (tRNA^(Sep)) in site specific incorporation ofphosphoserine into a protein or polypeptide. Typically. SepRSpreferentially aminoacylates tRNA^(Sep) with O-phosphoserine and thetRNA^(Sep) recognizes at least one codon such as a stop codon. Due tothe negative charge of the phosphoserine, Sep-tRNA^(Sep) does not bindelongation factor Tu (EF-Tu). However, the disclosed EF-Sep proteins canbind Sep-tRNA^(Sep) and protect Sep-tRNA^(Sep) from deacylation.

In some embodiments, EF-Sep is a mutant form of bacterial EF-Tu having amutation at one or more of amino acid residues corresponding to His67,Asp216, Glu217, Phe219, Thr229, and Asn274 in E. coli EF-Tu, which arelocated in the amino acid binding pocket for aminoacylated tRNA. In someembodiment, EF-Sep is a mutant form of eukaryotic elongation factor 1A(eEF1A) with mutations in positions equivalent to bacterial counterpart.

In preferred embodiments, EF-Sep has the amino acid sequence SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or a conservative variantthereof. For example, in some embodiments, the nucleic acid sequenceencoding EF-Sep has the nucleic acid sequence SEQ ID NO:5, SEQ ID NO:6.SEQ ID NO:7, SEQ ID NO:8, or a conservative variant thereof.

C. Variants

Also disclosed are variants of the disclosed proteins andpolynucleotides that include conservative substitutions, additions, anddeletions therein not affecting the structure or function. For example,biologically active sequence variants of tRNA^(Sep), SepRS, and EF-Sepand in vitro generated covalent derivatives of tRNA^(Sep), SepRS, andEF-Sep that demonstrate tRNA^(Sep), SepRS, and EF-Sep activity aredisclosed.

Various types of mutagenesis can be used to modify a nucleic acid. Theyinclude, but are not limited to, site-directed, random pointmutagenesis, homologous recombination (DNA shuffling), mutagenesis usinguracil containing templates, oligonucleotide-directed mutagenesis,phosphorothioate-modified DNA mutagenesis, and mutagenesis using methodssuch as gapped duplex DNA. Additional suitable methods include pointmismatch repair, mutagenesis using repair-deficient host strains,restriction-selection and restriction-purification, deletionmutagenesis, mutagenesis by total gene synthesis and double-strand breakrepair.

Sequence variants of tRNA^(Sep), SepRS, and EF-Sep fall into one or moreof three classes: substitutional, insertional and/or deletionalvariants. Sequence variants of tRNA^(Sep) include nucleotide variants,while sequence variants of SepRS and EF-Sep include nucleotide and/oramino acid variants. Insertions include amino and/or carboxyl terminalfusions as well as intrasequence insertions of single or multipleresidues. tRNA^(Sep), SepRS, and EF-Sep include, for example, hybrids ofmature tRNA^(Sep), SepRS, and EF-Sep with nucleotides or polypeptidesthat are homologous with tRNA^(Sep), SepRS, and EF-Sep. tRNA^(Sep),SepRS, and EF-Sep also include hybrids of tRNA^(Sep), SepRS, and EF-Sepwith nucleotides or polypeptides homologous to the host cell but not totRNA^(Sep), SepRS, and EF-Sep, as well as nucleotides or polypeptidesheterologous to both the host cell and tRNA^(Sep), SepRS, and EF-Sep.Fusions include amino or carboxy terminal fusions with eitherprokaryotic nucleotides or peptides or signal peptides of prokaryotic,yeast, viral or host cell signal sequences.

Insertions can also be introduced within the mature coding sequence oftRNA^(Sep), SepRS, and EF-Sep. These, however, ordinarily will besmaller insertions than those of amino or carboxyl terminal fusions, onthe order of one to four residues. Insertional sequence variants oftRNA^(Sep), SepRS, and EF-Sep are those in which one or more residuesare introduced into a predetermined site in the target tRNA^(Sep),SepRS, and EF-Sep.

Deletion variants are characterized by the removal of one or morenucleotides or amino acid residues from the tRNA^(Sep), SepRS, andEF-Sep sequence. For SepRS and EF-Sep, deletions or substitutions ofcysteine or other labile residues may be desirable, for example inincreasing the oxidative stability or selecting the preferred disulfidebond arrangement of SepRS or EF-Sep. Deletions or substitutions ofpotential proteolysis sites, e.g. Arg Arg, are accomplished, forexample, by deleting one of the basic residues or substituting one byglutaminyl or histidyl residues. Variants ordinarily are prepared bysite specific mutagenesis of nucleotides in the DNA encoding thetRNA^(Sep), SepRS, and EF-Sep, thereby producing DNA encoding thevariant, and thereafter expressing the DNA in recombinant cell culture.Variant tRNA^(Sep), SepRS, and EF-Sep fragments may also be prepared byin vitro synthesis. The variants typically exhibit the same qualitativebiological activity as the naturally-occurring analogue, althoughvariants also are selected in order to modify the characteristics oftRNA^(Sep), SepRS, and EF-Sep.

Substitutional variants are those in which at least one residue sequencehas been removed and a different residue inserted in its place. Owing tothe degeneracy of the genetic code, “silent substitutions” (i.e.,substitutions in a nucleic acid sequence which do not result in analteration in an encoded polypeptide) are an implied feature of everynucleic acid sequence which encodes an amino acid. Similarly,conservative amino acid substitutions are also readily identified. Suchconservative variations are a feature of each disclosed sequence. Thesubstitutions which in general are expected to produce the greatestchanges in SepRS or EF-Sep protein properties are those in which (a) ahydrophilic residue, e.g. seryl or threonyl, is substituted for (or by)a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl oralanyl; (b) a cysteine or proline is substituted for (or by) any otherresidue; (c) a residue having an electropositive side chain, e.g.,lysyl, arginyl, or histidyl, is substituted for (or by) anelectronegative residue, e.g., glutamyl or aspartyl; or (d) a residuehaving a bulky side chain, e.g., phenylalanine, is substituted for (orby) one not having a side chain, e.g., glycine.

While the site for introducing a nucleotide or amino acid sequencevariation is predetermined, the mutation per se need not bepredetermined. For example, in order to optimize the performance of amutation at a given site, random mutagenesis may be conducted at thetarget codon or region and the expressed tRNA^(Sep), SepRS, and EF-Sepvariants screened for the optimal combination of desired activity.Techniques for making substitution mutations at predetermined sites inDNA having a known sequence are well known.

Substitutions are typically of single residues; insertions usually willbe on the order of about from 1 to 10 residues; and deletions will rangeabout from 1 to 30 residues. Substitutions, deletion, insertions or anycombination thereof may be combined to arrive at a final construct. Themutations that will be made in the DNA encoding the variant SepRS andEF-Sep must not place the sequence out of reading frame and preferablywill not create complementary regions that could produce secondary mRNAstructure.

A DNA isolate is understood to mean chemically synthesized DNA, cDNA orgenomic DNA with or without the 3′ and/or 5′ flanking regions. DNAencoding tRNA^(Sep), SepRS, and EF-Sep can be obtained from othersources than Methanocaldococcus jannaschii by screening a cDNA libraryfrom cells containing mRNA using hybridization with labeled DNA encodingMethanocaldococcus jannaschii tRNA^(Sep), SepRS, and EF-Sep, orfragments thereof (usually, greater than 10 bp).

The precise percentage of similarity between sequences that is useful inestablishing sequence identity varies with the nucleic acid and proteinat issue, but as little as 25% sequence similarity is routinely used toestablish sequence identity. Higher levels of sequence similarity, e.g.,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more can also be usedto establish sequence identity. Methods for determining sequencesimilarity percentages (e.g., BLASTP and BLASTN using defaultparameters) are generally available.

Alignment of sequences for comparison can be conducted by manywell-known methods in the art, for example, by the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by thehomology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443(1970), by the search for similarity method of Pearson & Lipman, Proc.Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), by the Gibbs sampling method (Chatterji and Pachter, JComput Biol. 12(6):599-608 (2005)), by PSI-BLAST-ISS (Margelevicius andVenclovas, BMC Bioinformatics 21; 6:185 (2005)), or by visualinspection. One algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information (www.ncbi.nlm.nib.gov).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysts of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

D. Expression or Translation Systems

Also disclosed are expression or translation systems for incorporatephosphoserine into a growing polypeptide chain (protein). Components ofa translation system generally include amino acids, ribosomes, tRNAs,synthetases, and mRNA. The disclosed tRNA^(Sep), SepRS, and EF-Sep canbe added to a translation system, in vivo or in vitro to incorporatephosphoserine into a protein.

In some embodiments, a cell-based (in vivo) expression system is used.In these embodiments, nucleic acids encoding one or more of tRNA^(Sep),SepRS, and EF-Sep are delivered to cells under conditions suitable fortranslation and or transcription of tRNA^(Sep), SepRS, EF-Sep, or acombination thereof. The cells can in some embodiments be prokaryotic,e.g., an E. coli cell, or eukaryotic, e.g., a yeast, mammalian, plant,or insect or cells thereof.

In some embodiments, a cell-free (in vitro) expression system is used.The most frequently used cell-free translation systems involve extractscontaining all the macromolecular components (70 S or 80 S ribosomes,tRNAs, aminoacyl-tRNA synthetases, initiation, elongation andtermination factors, etc.) required for translation of exogenous RNA. Toensure efficient translation, each extract is supplemented with aminoacids, energy sources (ATP, GTP), energy regenerating systems (creatinephosphate and creatine phosphokinase for eukaryotic systems, andphosphoenol pyruvate and pyruvate kinase for the E. coli lysate), andother co-factors (Mg²⁺, K⁻etc.).

i) Promoters and Enhancers

Nucleic acids that are delivered to cells typically contain expressioncontrolling systems. For example, the inserted genes in viral andretroviral systems usually contain promoters, and/or enhancers to helpcontrol the expression of the desired gene product. A promoter isgenerally a sequence or sequences of DNA that function when in arelatively fixed location in regard to the transcription start site. Apromoter contains core elements required for basic interaction of RNApolymerase and transcription factors, and may contain upstream elementsand response elements.

Therefore, also disclosed is a polynucleotide encoding one or more oftRNA^(Sep), SepRS, and EF-Sep, operably linked to an expression controlsequence.

Suitable promoters are generally obtained from viral genomes (e.g.,polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-Bvirus, and cytomegalovirus) or heterologous mammalian genes (e.g. betaactin promoter). Enhancer generally refers to a sequence of DNA thatfunctions at no fixed distance from the transcription start site and canbe either 5′ or 3′ to the transcription unit. Furthermore, enhancers canbe within an intron as well as within the coding sequence itself. Theyare usually between 10 and 300 bp in length, and they function in cis.Enhancers function to increase transcription from nearby promoters.Enhancers also often contain response elements that mediate theregulation of transcription. Many enhancer sequences are now known frommammalian genes (globin, elastase, albumin, α-fetoprotein and insulin).However, enhancer from a eukaryotic cell virus are preferably used torgeneral expression. Suitable examples include the SV40 enhancer on thelate side of the replication origin, the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

In certain embodiments the promoter and/or enhancer region can act as aconstitutive promoter and/or enhancer to maximize expression of theregion of the transcription unit to be transcribed. In certainconstructs the promoter and/or enhancer region is active in aileukaryotic cell types, even if it is only expressed in a particular typeof cell at a particular time. A preferred promoter of this type is theCMV promoter. In other embodiments, the promoter and/or enhancer istissue or cell specific.

In certain embodiments the promoter and/or enhancer region is inducible.Induction can occur, e.g., as the result of a physiologic response, aresponse to outside signals, or as the result of artificialmanipulation. Such promoters are well known to those of skill in theart. For example, in some embodiments, the promotor and/or enhancer maybe specifically activated either by light or specific chemical eventswhich trigger their function. Systems can be regulated by reagents suchas tetracycline and dexamethasone. There are also ways to enhance viralvector gene expression by exposure to irradiation, such as gammairradiation, or alkylating chemotherapy drugs.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human or nucleated cells) may also contain sequencesnecessary for the termination of transcription which may affect mRNAexpression. These regions are transcribed as polyadenylated segments inthe untranslated portion of the mRNA encoding tissue factor protein. The3′ untranslated regions also include transcription termination sites. Itis preferred that the transcription unit also contains a polyadenylationregion. One benefit of this region is that it increases the likelihoodthat the transcribed unit will be processed and transported like mRNA.The identification and use of polyadenylation signals in expressionconstructs is well established. It is preferred that homologouspolyadenylation signals be used in the transgene constructs.

ii) Cell Delivery Systems

There are a number of compositions and methods which can be used todeliver nucleic acids to cells, either in vitro or in vivo. Thesemethods and compositions can largely be broken down into two classes:viral based delivery systems and non-viral based delivery systems. Forexample, nucleic acids can be delivered through a number of directdelivery systems such as electroporation, lipofection, calcium phosphateprecipitation, plasmids, viral vectors, viral nucleic acids, phagenucleic acids, phages, cosmids, or via transfer of genetic material incells or carriers such as cationic liposomes. Appropriate means fortransfection, including viral vectors, chemical transfectants, orphysico-mechanical methods such as electroporation and direct diffusionof DNA, are well known in the art and readily adaptable for use with thecompositions and methods described herein.

Transfer vectors can be any nucleotide construction used to delivergenetic material into cells. In some embodiments the vectors are derivedfrom either a virus or a retrovirus. Viral vectors include, for example,Adenovirus, Adeno-associated virus, Herpes virus, Vaccinia virus, Poliovirus, AIDS virus, neuronal trophic virus, Sindbis and other RNAviruses, including these viruses with the HIV backbone.

Typically, viral vectors contain nonstructural early genes, structurallate genes, an RNA polymerase III transcript, inverted terminal repeatsnecessary for replication and encapsidation, and promoters to controlthe transcription and replication of the viral genome. When engineeredas vectors, viruses typically have one or more of the early genesremoved and a gene or gene/promotor cassette is inserted into the viralgenome in place of the removed viral DNA. The necessary functions of theremoved early genes are typically supplied by cell lines which have beenengineered to express the gene products of the early genes in trans.

Nucleic acids can also be delivered through electroporation,sonoporation, lipofection, or calcium phosphate precipitation.Lipofection involves the use liposomes, including cationic liposomes(e.g., DOTMA, DOPE, DC-cholesterol) and anionic liposomes, to deliverygenetic material to a cell. Commercially available liposome preparationsinclude LIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.),SUPERFECT (Qiagen, Inc. Hilden, Germany), and TRANSFECTAM (PromegaBiotec, Inc,. Madison, Wis.).

Nucleic acids that are delivered to cells which are to be integratedinto the host cell genome, typically contain integration sequences.These sequences are often viral related sequences, particularly whenviral based systems are used. These viral intergration systems can alsobe incorporated into nucleic acids which are to be delivered using anon-nucleic acid based system of deliver, such as a liposome, so thatthe nucleic acid contained in the delivery system can be come integratedinto the host genome. Techniques for integration of genetic materialinto a host genome are also known and include, for example, systemsdesigned to promote homologous recombination with the host genome. Thesesystems typically rely on sequence flanking the nucleic acid to beexpressed that has enough homology with a target sequence within thehost cell genome that recombination between the vector nucleic acid andthe target nucleic acid takes place, causing the delivered nucleic acidto be integrated into the host genome. These systems and the methodsnecessary to promote homologous recombination are known to those ofskill in the art.

iii) Markers

The vectors used to deliver the disclosed nucleic acids to cells canfurther include nucleic acid sequence encoding a marker product. Thismarker product is used to determine if the gene has been delivered tothe cell and once delivered is being expressed. In some embodiments themarker is a detectable label. Exemplary labels include the E. coli lacZgene, which encodes β-galactosidase, and green fluorescent protein(GFP).

In some embodiments the marker may be a selectable marker. Examples ofsuitable selectable markers for mammalian cells are dihydrofolatereductase (DHFR), thymidine kinase, neomycin, neomycin analog G418,hydromycin, and puromycin. When such selectable markers are successfullytransferred into a mammalian host cell, the transformed mammalian hostcell can survive if placed under selective pressure. There are twowidely used distinct categories of selective regimes. The first categoryis based on a cell's metabolism and the use of a mutant cell line whichlacks the ability to grow independent of a supplemented media. Thesecond category is dominant selection which refers to a selection schemeused in any cell type and does not require the use of a mutant cellline. These schemes typically use a drug to arrest growth of a hostcell. Those cells which have a novel gene would express a proteinconveying drug resistance and would survive the selection.

III. Methods A. Site-Specific Phosphorylation of Proteins

Methods for incorporating phosphoserine into polypeptides are disclosed.The method involves the use of tRNA^(Sep), SepRS, and EF-Sep in thetranslation process for a target polypeptide from mRNA. SepRSpreferentially aminoacylates tRNA^(Sep) with O-phosphoserine. Theresulting Sep-tRNA^(Sep) recognizes at least one codon in the mRNA forthe target protein, such as a stop codon. EF-Sep mediates the entry ofthe Sep-tRNA^(Sep) into a free site of the ribosome. If thecodon-anticodon pairing is correct, EF-Sep hydrolyzes guanosinetriphosphate (GTP) into guanosine diphosphate (GDP) and inorganicphosphate, and changes in conformation to dissociate from the tRNAmolecule. The Sep-tRNA^(Sep) then fully enters the A site, where itsamino acid is brought near the P site's polypeptide and the ribosomecatalyzes the covalent transfer of the amino acid onto the polypeptide.

In preferred embodiments, the tRNA^(Sep) is a tRNA^(Cys) from amethanogenic archaea, such as Methanocaldococcus jannaschii, containinga mutation (e.g., C20U) that improves aminoacylation of the tRNA bySepRS without affecting CysRS recognition. In some embodiments, thetRNA^(Sep) contains an anticodon that binds a codon other than a Cyscodon, such as a stop codon. In some embodiments, the tRNA^(Sep) isencoded the nucleic acid sequence SEQ ID NO:41, or a conservativevariant thereof.

In some embodiments, the SepRS is any tRNA synthetase thatpreferentially aminoacylates tRNA^(Sep) with a phosphoserine. Inpreferred embodiments, the SepRS is a tRNA synthetase from amethanogenic archaea, such as Methanococcus maripaludis orMethanocaldococcus jannaschii. In some embodiments, the SepRS has theamino acid sequence SEQ ID NO:43 or 46, or a conservative variantthereof.

In some embodiments, the EF-Sep is any elongation factor protein thatbinds Sep-tRNA^(Sep) and catalyzes the covalent transfer of thephosphoserine amino acid onto the polypeptide. EF-Sep proteins can bindSep-tRNA^(Sep) and can preferably protect Sep-tRNA^(Sep) fromdeacylation. In some embodiments, EF-Sep is a mutant form of bacterialEF-Tu having a mutation at one or more of amino acid residuescorresponding to His67, Asp216, Glu217, Phe219, Thr229, and Asn274 in E.coli EF-Tu, which are located in the amino acid binding pocket foraminoacylated tRNA. In some embodiments, EF-Sep has the amino acidsequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or aconservative variant thereof. In some embodiments, EF-Sep is a mutantform of eukaryotic elongation factor 1A (eEF1A) with mutations inpositions equivalent to bacterial counterpart.

i) In Vitro Transcription/Translation

In one embodiment, the nucleic acids encoding tRNA^(Sep) and SepRSactivity are synthesized prior to translation of the target protein andare used to incorporate phosphoserine into a target protein in acell-free (in vitro) protein synthesis system.

In vitro protein synthesis systems involve the use crude extractscontaining all the macromolecular components (70 S or 80 S ribosomes.tRNAs, aminoacyl-tRNA synthetases, initiation, elongation andtermination factors, etc.) required for translation of exogenous RNA.For the current method, the tRNAs, aminoacyl-tRNA synthetases, andelongation factors in the crude extract are supplemented withtRNA^(Sep), SepRS, and EF-Sep. To ensure efficient translation, eachextract must be supplemented with amino acids, energy sources (ATP,GTP), energy regenerating systems (creatine phosphate and creatinephosphokinase for eukaryotic systems, and phosphoenol pyruvate andpyruvate kinase for the E. coli lysate), and other co-factors (Mg2+, K+,etc.).

In vitro protein synthesis does not depend on having a polyadenylatedRNA, but if having a poly(A) tail is essential for some other purpose, avector may be used that has a stretch of about 100 A residuesincorporated into the polylinker region. That way, the poly(A) tail is“built in” by the synthetic method. In addition, eukaryotic ribosomesread RNAs that have a 5′ methyl guanosine cap more efficiently. RNA capscan be incorporated by initiation of transcription using a capped baseanalogue, or adding a cap in a separate in vitro reactionpost-transcriptionally.

Suitable in vitro transcription/translation systems include, but are notlimited to, the rabbit reticulocyte system, the E. coli S-30transcription-translation system, the wheat germ based translationalsystem. Combined transcription/translation systems are available, inwhich both phage RNA polymerases (such as T7 or SP6) and eukaryoticribosomes are present. One example of a kit is the TNT® system fromPromega Corporation.

ii) In Vivo Methods

Host cells and organisms can also incorporate phosphoserine intoproteins or polypeptides via nucleic acids encoding tRNA^(Sep), SepRS,and EF-Sep. Nucleic acids encoding tRNA Sep, SepRS, and EF-Sep, operablylinked to one or more expression control sequences are introduced intocells or organisms using a cell delivery system. These cells alsocontain a gene encoding the target protein operably linked to anexpression control sequence.

Suitable organisms include, but are not limited to, microorganisms suchas bacteria transformed with recombinant bacteriophage, plasmid, orcosmid DNA expression vectors; yeast transformed with yeast expressionvectors; insect cell systems infected with viral expression vectors(e.g., baculovirus); plant cell systems transformed with viralexpression vectors (e.g., cauliflower mosaic virus, CaMV, or tobaccomosaic virus, TMV) or with bacterial expression vectors (e.g., Ti orpBR322 plasmids); or animal cell systems.

It will be understood by one of ordinary skill in the art thatregardless of the system used (i.e. in vitro or in vivo), expression ofgenes encoding tRNA^(Sep), SepRS, and EF-Sep activity will result insite specific incorporation of phosphoserine into the targetpolypeptides or proteins that are translated in the system. Host cellsare genetically engineered (e.g., transformed, transduced ortransfected) with the vectors encoding tRNA^(Sep), SepRS, and EF-Sep,which can be, for example, a cloning vector or an expression vector. Thevector can be, for example, in the form of a plasmid, a bacterium, avirus, a naked polynucleotide, or a conjugated polynucleotide. Thevectors are introduced into cells and/or microorganisms by standardmethods including electroporation, infection by viral vectors, highvelocity ballistic penetration by small particles with the nucleic acideither within the matrix of small beads or particles, or on the surface.Such vectors can optionally contain one or more promoter. A “promoter”as used herein is a DNA regulatory region capable of initiatingtranscription of a gene of interest.

Kits are commercially available for the purification of plasmids frombacteria, (see, e.g., GFX™ Micro Plasmid Prep Kit from GE Healthcare;Strataprep® Plasmid Miniprep Kit and StrataPrep® EF Plasmid Midiprep Kitfrom Stratagene; Gen Elute™ HP Plasmid Midiprep and Maxiprep Kits fromSigma-Aldrich, and, Qiagen plasmid prep kits and QIAfilter™ kits fromQiagen). The isolated and purified plasmids are then further manipulatedto produce other plasmids, used to transfect cells or incorporated intorelated vectors to infect organisms. Typical vectors containtranscription and translation terminators, transcription and translationinitiation sequences, and promoters useful for regulation of theexpression of the particular target nucleic acid. The vectors optionallycomprise generic expression cassettes containing at least oneindependent terminator sequence, sequences permitting replication of thecassette in eukaryotes, or prokaryotes, or both, (e.g., shuttle vectors)and selection markers for both prokaryotic and eukaryotic systems.

Prokaryotes useful as host cells include, but are not limited to, gramnegative or gram positive organisms such as E. coli or Bacilli. In aprokaryotic host cell, a polypeptide may include an N-terminalmethionine residue to facilitate expression of the recombinantpolypeptide in the prokaryotic host cell. The N-terminal Met may becleaved from the expressed recombinant polypeptide. Promoter sequencescommonly used for recombinant prokaryotic host cell expression vectorsinclude lactamase and the lactose promoter system.

Expression vectors for use in prokaryotic host cells generally compriseone or more phenotypic selectable marker genes. A phenotypic selectablemarker gene is, for example, a gene encoding a protein that confersantibiotic resistance or that supplies an autotrophic requirement.Examples of useful expression vectors for prokaryotic host cells includethose derived from commercially available plasmids such as the cloningvector pBR322 (ATCC 37017). pBR322 contains genes for ampicillin andtetracycline resistance and thus provides simple means for identifyingtransformed cells. To construct an expression vector using pBR322, anappropriate promoter and a DNA sequence are inserted into the pBR322vector. Other commercially available vectors include, for example, T7expression vectors from Invitrogen, pET vectors from Novagen and pALTER®vectors and PinPoint® vectors from Promega Corporation.

Yeasts useful as host cells include, but are not limited to, those fromthe genus Saccharomyces, Pichia, K. Actinomycetes and Kluyveromyces.Yeast vectors will often contain an origin of replication sequence, anautonomously replicating sequence (ARS), a promoter region, sequencesfor polyadenylation, sequences tor transcription termination, and aselectable marker gene. Suitable promoter sequences for yeast vectorsinclude, among others, promoters for metallothionein, 3-phosphoglyceratekinase (Hitzeman et al., Biol. Chem. 255:2073, (1980)) or otherglycolytic enzymes (Holland et al., Biochem. 17:4900, (1978)) such asenolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. Other suitable vectors andpromoters for use in yeast expression are further described m Fleer etal., Gene, 107:285-195 (1991), in Li, et al., Lett Appl Microbiol.40(5):347-52 (2005), Jansen, et al., Gene 344:43-51 (2005) and Daly andHearn, J. Mol. Recognit. 18(2):119-38 (2005). Other suitable promotersand vectors for yeast and yeast transformation protocols are well knownin the art.

Mammalian or insect host cell culture systems well known in the art canalso be employed to express recombinant tRNA^(Sep), SepRS, and EF-Sepfor producing proteins or polypeptides containing phosphoserine.Commonly used promoter sequences and enhancer sequences are derived fromPolyoma virus, Adenovirus 2, Simian Virus 40 (SV40), and humancytomegalovirus. DNA sequences derived from the SV40 viral genome may beused to provide other genetic elements for expression of a structuralgene sequence in a mammalian host cell, e.g., SV40 origin, early andlate promoter, enhancer, splice, and polyadenylation sites. Viral earlyand late promoters are particularly useful because both are easilyobtained from a viral genome as a fragment which may also contain aviral origin of replication. Exemplary expression vectors for use inmammalian host cells are well known in the art.

B. Purifying Proteins Containing Phosphoserine

Proteins or polypeptides containing phosphoserine can be purified,either partially or substantially to homogeneity, according to standardprocedures known to and used by those of skill in the art including, butnot limited to, ammonium sulfate or ethanol precipitation, acid or baseextraction, column chromatography, affinity column chromatography, anionor cation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, hydroxylapatite chromatography,lectin chromatography, and gel electrophoresis. Protein refolding stepscan be used, as desired, in making correctly folded mature proteins.High performance liquid chromatography (HPLC), affinity chromatographyor other suitable methods can be employed in final purification stepswhere high purity is desired. In one embodiment, antibodies made againstproteins containing phosphoserine are used as purification reagents,e.g., for affinity-based purification of proteins containingphosphoserine. Once purified, partially or to homogeneity, as desired,the polypeptides may be used as assay components, therapeutic reagents,immunogens for antibody production, etc.

Those of skill in the art will recognize that, after synthesis,expression and/or purification, proteins can possess conformationsdifferent from the desired conformations of the relevant polypeptides.For example, polypeptides produced by prokaryotic systems often areoptimized by exposure to chaotropic agents to achieve proper folding.During purification from lysates derived from E. coli, the expressedprotein is optionally denatured and then renatured. This is accomplishedby solubilizing the proteins in a chaotropic agent such its guanidineHCl.

It is occasionally desirable to denature and reduce expressedpolypeptides and then to cause the polypeptides to re-fold into thepreferred conformation. For example, guanidine, urea, DTT, DTE, and/or achaperonin can be added to a translation product of interest. Methods ofreducing, denaturing and renaturing proteins are well known to those ofskill in the art Refolding reagents can be flowed or otherwise movedinto contact with the one or more polypeptide or other expressionproduct, or vice-versa.

C. Using Phosphoserine Containing Peptides

Proteins or polypeptides containing phosphoserine and antibodies thatbind to such proteins produced by the methods described herein can beused for research involving phosphoproteins such as the study ofkinases, phosphotases, and target proteins in signal transductionpathways. Proteins or polypeptides containing phosphoserine produced bythe methods described herein can also be used for antibody production,protein array manufacture and development of cell-based screens for newdrug discovery.

IV. Kits

Kits for producing polypeptides and/or proteins containing phosphoserineare also provided. For example, a kit for producing a protein thatcontains phosphoserine in a cell is provided, where the kit includes apolynucleotide sequence encoding tRNA^(Sep), a polynucleotide sequenceencoding SepRS, and a polynucleotide sequence encoding EF-Sep. In oneembodiment, the kit further includes phosphoserine. In anotherembodiment, the kit further comprises instructional materials torproducing the protein. In another embodiment, a kit for producing aprotein that contains phosphoserine in vitro is provided, where the kitincludes a polynucleotide sequence encoding tRNA^(Sep), a polynucleotidesequence encoding SepRS, a polynucleotide sequence encoding EF-Sep, andphsophoserine. In another embodiment, the kit further comprisesinstructional materials for producing the protein in vitro.

The present invention will be further understood by reference to thefollowing non-limiting examples.

EXAMPLES Example 1: SepRS and tRNA^(Sep) are an Orthogonal Pair in E.coli Materials and Methods Constructions of Strains

To prevent possible enzymatic dephosphorylation of O-phospho-L-serine(Sep) in vivo, the gene encoding phosphoserine phosphatase (serB), whichcatalyzes the last step in serine biosynthesis, was deleted fromEscherichia coli strains Top 10 and BL21. Markerless gene deletions werecarried out using a λ-red and FLP recombinase-based gene knockoutstrategy as described by Datsenko K A, et al. Proc Natl Acad Sci USA.97:6640 (2000). E. coli strains Top 10ΔserB and BL21ΔserB were used forEF-Tu library construction and MEK1 expression experiments.

Construction of Plasmids

To construct plasmid pSepT, the full-length gene encoding tRNA^(Sep) wasconstructed from overlapping oligonucleotides and ligated immediatelydownstream of the lpp promoter in pTECH (Bunjun S, et al. Proc Natl AcadSci USA. 97:12997 (2000)) using EcoRI and BamHI restriction sites.pCysT, encoding the wild type tRNA^(Cys) gene from Methanocaldococcusjannaschii was constructed in the same way.

The gene fragment encoding β-lactamase was PCR-amplified from plasmidpUC18 using primers PBLAF (5′-TGC GCA ATG CGG CCG CCC GTA GCG CCG ATGGTA GTG T-3′, SEQ ID NO:9) and PBLAR (5′-ACA CGG AGA TCT CTA AAG TAT ATATGA GTA AAC-3′, SEQ ID NO:10), and ligated with a NotI and BglIIdigested PCR product which was constructed from pSepT using primersPSEPF (5′-TGC GCA ATG CGG CCG CCC GGG TCG AAT TTG CTT TCG A-3′, SEQ IDNO:11) and PSEPR (5′-ACA CGG AGA TCT ATG CCC CGC GCC CAC CGG AAG-3′, SEQID NO:12).

pKD was derived from pKK223-3 (Pharmacia). The ampicillin resistancegene was replaced with a kanamycin resistance gene by combining two PCRproducts generated from pKK223-3 and pET28a. The following PCR primerswere used: PKF (5′-TGC AGCA ATG CGG CCG CTT TCA CCG TCA TCA CCG AAAC-3′, SEQ ID NO:13) and PKR (5′-GGG ACG CTA GCA AAC AAA AAG AGT TTG TAGAA-3′, SEQ ID NO:14) for pKK223-3 amplification and PKNF (5′-GGG ACG CTAGCT TTT CTC TGG TCC CGC CGC AT-3′, SEQ ID NO:15) and PKNR (5′-TGC GCAATG CGG CCG CGG TGG CAC TTT TCG GGG AAA T-3′, SEQ ID NO:16) for Kan^(R)gene amplification.

The original multiple cloning site (MCS; NcoI-EcoRI-SacI) was modifiedby adding an additional ribosome binding site (RBS) and a second MCS(NdeI-BamHI-SalI-HindIII), thus enabling simultaneous protein expressionfront two genes, both under the control of the same tac promoter. TheMethanococcus maripaludis SepRS gene was cloned into pKD using NcoI andSacI sites to produce pKD-SepRS. The E. coli EE-Tu gene (tufB) wasligated into pKD-SepRS using BamHI and SalI sites resulting inpKD-SepRS-EFTu. The M. maripaludis pscS gene encoding SepCysS was clonedinto pKD-SepRS using BamHI and SalI sites to produce pKD-SepRS-SepCysSand the M. maripaludis CysRS gene was cloned into pKD using EcoRI andSalI to yield pKD-CysRS.

pCAT112TAG-SepT was created from pACYC184. The gene encodingchloramphenicol acetyltransferae (CAT) was modified by quickchangemutagenesis to introduce an amber stop codon at position Asp112 (Wang L,et al. Science 292:498 (2001)). The resulting plasmid was PCR amplifiedusing primers PBLAF (5′-TGC GCA ATG CGG CCG CCC GTA GCG CCG ATG GTA GTGT-3′, SEQ ID NO:9) and PBLAR (5′-ACA CGG AGA TCT CTA AAG TAT ATA TGA GTAAAC-3′, SEQ ID NO:10) and ligated with a PCR product containing atRNA^(Sep) expression cassette from pSepT, created with primers TSEPF(5′-GCA TGC GCC GCC AGC TGT TGC CCG TCT CGC-3′, SEQ ID NO:17) and TSEPR(5′-GCA TAG ATC TTC AGC TGG CGA AAG GGG GAT G-3′, SEQ ID NO:18).

Plasmid pCcdB was created by adding a ccdB gene under the control of alac promoter into pTECH using NotI and BglII sites (Wang L, et al.Science 292:498 (2001)). Two amber stop codons were introduced atpositions 13 and 44 based on the crystal structure and mutagenesis studyof the CcdB protein (Bernard. P., et al. Gene 148:71 (1994); Bajaj K. etal. Proc Natl Acad Sci USA 102:16221 (2005)).

Plasmid pL11C-SepT encodes tRNA^(Sep) and the C-terminal domain of theribosomal protein L11 under control of lpp promoters. Part of the rplKgene was PCR amplified from genomic E. coli DNA using primers L11C-F(5′-GGA ATT CCA TAT GAC CAA GAC CCC GCC GGC AGC AGT T-3′, SEQ ID NO:38)and L11C-R (5′-AGG CGC GCC TTA GTC CTC CAC TAC-3′, SEQ ID NO:39). ThePCR product was digested with NdeI and AscI and was ligated into NdeIand AscI digested pMYO127TAG-SepT to replace the myoglobin gene.

To construct pMAL-EFTu and pMAL-EFSep E. coli tufB, or the gene encodingEF-Sep, respectively, were cloned between the NdeI and BamHI sites inthe pET20b plasmid (Novaven) to add a C-terminal His₆ tag. This fusionconstruct was then PCR-amplified using primers adding MfeI and PstIrestriction sites. The PCR product was cloned in-frame between EcoRI andPstI in pMAL-c2x (New England Biolabs) to add an N-terminal maltosebinding protein (MBP) tag.

Aminoacylation of tRNA and EF-Sep Binding Assays

In vitro transcript of Methanocaldococcus jannaschii tRNA ^(Cys) wasprepared and acylated with [¹⁴C]Sep (55 mCi/mmol) using recombinantMethanococcus maripaludis SepRS as described previously by Hohn M J, etal. Proc Natl Acad Sci USA. 2006 Nov. 28; 103(48); 18095-100.Sep-tRNA^(Cys) was phenol/chlorophorm extracted, and the aqueous phasewas passed over Sephadex G25 Microspin columns(GE Healthcare)equilibrated with water.

Protection of Sep-tRNA^(Cys) by EF-Tu was assayed as described earlierwith slight modifications (Ling J, et al. RNA. 2007 November;13(11);1881 -6.). Briefly, EF-Tu or EF-Sep (both purified as maltosebinding protein fusion proteins) were activated for 20 min. at 37°C. inbuffer containing 100 mM Tris-HCl (pH 8.2), 120 mM NH₄Cl, 7 mM MgCl₂, 5mM DTT, 5 mM phosphoenolpyruvate, 1.5 mM GTP, and 0.12 μg/μl pyruvatekinase. Hydrolysis of 2 μM [¹⁴C]Sep-tRNA^(Cys) was then monitored at25°C. in the presence of 40 μM EF-Tu (wt), EF-Sep, or BSA, respectively.Aliquots were taken from the reaction mix at indicated time points andspotted on 3 MM filter discs presoaked with 10% trichloroacetic acid.Filters were washed with 5% trichloroacetic acid, dried, andradioactivity was measured by liquid scintillation counting.

Results

The Sep-insertion strategy was based on the discovery that mostmethanogens form Cys-tRNA^(Cys) by an unusual pathway required forcysteine synthesis in these archaea (Sauerwald A. et al. Science 307,1969 (2005)). In this route (FIG. 1A), tRNA^(Cys) first becomes acylatedwith O-phosphoserine (Sep) by O-phosphoseryl-tRNA synthetase (SepRS), anunusual aminoacyl-tRNA synthetase specific solely for the substrates Sepand tRNA^(Cys) (Hohn, M J., et al. Proc Natl Acad Sci USA 103, 18095(2006)). The resulting product Sep-tRNA^(Cys) is then converted toCys-tRNA^(Cys) by the enzyme SepCysS in the presence of a sulfur-donor(Sauerwald A. et al., Science 307, 1969 (2005)). The exclusiverecognition of Sep by SepRS was further confirmed by the structuralelucidation of this enzyme and by the biochemical analysis of itscatalytic site (Kamtekar S. et al., Proc Natl Acad Sci USA 104, 2620(2007); Fukunaga, R. et al., Nat Struct Mol Biol 14, 272 (2007)). Themolecular basis of Methanocaldococcus jannaschii (Mj) tRNA^(Cys)recognition by SepRS and CysRS from Methanococcus maripaludis (Mmp) wasalso explored, yielding the SepRS-specific tRNA identity elements (Hohn,M J., et al., Proc Natl Acad Sci USA 103, 18095 (2006)). Based on theseresults it was decided to test the applicability of Mj tRNA^(Cys) andMmp SepRS as an orthogonal pair for UAG-directed translationalincorporation of Sep into proteins expressed in Escherichia coli. Ascheme was sought for co-translational insertion of phosphoserine (Sep)into proteins in E. coli in response to the amber codon UAG. Methanogensutilize an aminoacyl-tRNA synthetase (SepRS) that acylates tRNA^(Cys)with Sep during the biosynthesis of Cys-tRNA^(Cys) (FIG. 1A).

A tRNA (tRNA^(Sep)) was designed that could be aminoacylated withphosphoserine (FIG. 1B). tRNA^(Sep) is a tRNA derived from Mj tRNA^(Cys)containing a C20U change that improves 2.5-fold the aminoacylation bySepRS without affecting CysRS recognition. In addition, tRNA^(Sep) wasmodified to be an amber suppressor by including two mutations in theanticodon (FIG. 1B).

Both tRNA^(Sep) and tRNA^(Cys) were overexpressed in E. coli. In vivoaminoacylation by Mmp SepRS showed (FIG. 1C) that the anticodon changelowered (to about 40%) the activity of tRNA^(Sep) when compared totRNA^(Cys). Total E. coli tRNA could not be charged with Sep (FIG. 1C).Based on these in vitro data, Mj tRNA^(Sep) and Mmp SepRS appear to bean orthogonal pair.

Efficient and selective addition of Sep to the E. coli geneticrepertoire requires exclusive interaction of SepRS with tRNA^(Sep) forSep-tRNA^(Sep) formation without interfering in the host translationsystem as well as a sufficient intracellular concentration of Sep. As E.coli has a Sep-compatible transporter (Wanner, B L. FEMS Microbiol Lett79, 133 (1992)). Sep (2 mM) was added to the growth medium, and theendogenous serB gene encoding phosphoserine phosphatase was deleted inthe E. coli test strain. To assess whether the Mj tRNA^(Sep)/Mmp SepRSpair is functional and orthogonal in E. coli, a suppression assay wasperformed that employed a chloramphenicol acetyltransferase (CAT) genewith a UAG stop codon at the permissive position 112 (wild-type aminoacid: Asp) to produce chloramphenicol (Cm) acetyltransferase: then cellsurvival was measured in the presence of Sep and varying amounts of Cm.The different IC₅₀ values (FIG. 2) relate to suppression efficiency(i.e., amount of CAT made dependent on the various transformed genes).When only tRNA^(Sep) is expressed (FIG. 2, second bar) Cm resistanceincreases about 3.3-fold over background (FIG. 2, first bar). Thus,tRNA^(Sep) can be aminoacylated to a certain degree by an unknown E.coli aminoacyl-tRNA synthetase (Gln is being incorporated at the amberstop codon). In contrast, simultaneous expression of tRNA^(Sep) andSepRS does not provide Cm resistance (FIG. 2. third bar). This mayindicate that SepRS can out-compete any endogenous aminoacyl-tRNAsynthetase and form Sep-tRNA^(Sep); however, this aminoacyl-tRNA is notdelivered to the ribosome or not accommodated on the ribosome. Providingadditional EF-Tu does not improve the result (FIG. 2, fifth bar).Co-expression of tRNA^(Sep), SepRS and SepCysS should result information of Sep-tRNA^(Sep) and subsequent SepCysS-mediated conversionto Cys-tRNA^(Sep) (A. Sauerwald et al., Science 307, 1969 (2005)).Indeed, a 2.3-fold increase in Cm resistance is observed (FIG. 2, sixthbar). This further supports the notion that while Sep-tRNA^(Sep) issynthesized, it cannot be used properly by the E. coli proteinbiosynthesis machinery. On the other hand, co-expression of tRNA^(Sep)and Mmp CysRS generates a 12.3-fold increase in Cm resistance (FIG. 2,eight bar), demonstrating that Cys-tRNA^(Sep) can be readily used foramber codon suppression in the CAT gene.

Given that EF-Tu is a component of quality control in protein synthesis(LaRiviere, F J., et al. Science 294, 165 (2001)), it is highlyplausible that Sep-tRNA^(Sep) may be rejected by EF-Tu in order not tointerfere with the complicated cellular mechanism of phosphoproteinproduction. Chemically synthesized Sep-tRNA^(Gln) was a poor substratefor in vitro protein synthesis (Rothman D M. et al., J Am Chem Soc 127,846 (2005)). tRNAs carrying negatively charged amino acids are boundpoorly by EF-Tu (Dale, T., et al. Biochemistry 43, 6159 (2004)), andmolecular dynamics simulations suggested that Sep-tRNA^(Cys) may not bebound by EF-Tu (Eargle, J., et al. J Mol Biol 377, 1382 (2008)). Thisassumption was tested in EF-Tu mediated Sep-tRNA hydrolysis protectionexperiments (J. Ling et al., Proc Natl Acad Sci USA 104, 15299 (2007)),and incubated recombinant E. coli EF-Tu with the Mj tRNA^(Cys) in vitrotranscript either acylated with [³⁵S]Cys or [¹⁴C]Sep at pH 8.2. WhileEF-Tu protected [³⁵S]Cys-tRNA^(Cys) from deacylation (FIG. 5A).Sep-tRNA^(Cys) was significantly deacylated irrespective of the presenceof EF-Tu (FIG. 3 and FIG. 5B). Thus, insufficient binding ofSep-tRNA^(Sep) to EF-Tu may explain the lack of Sep insertion intoprotein.

Example 2: Development of EF-Sep Materials and Methods LibraryConstruction and Selection of Sep-tRNA Specific EF-Tu

Six residues, His67, Asp216, Glu217, Phe2l9, Thr229, and Asn274, locatedin the amino acid binding pocket of the E. coli elongation factor EF-Tuwere selected for randomization based on the crystal structure of the E.coli EF-Tu:Phe-tRNA^(Phe) complex (protein data base accession number1OB2). Multiple rounds of overlap PCR were carried out to incorporaterandom codons (NNK) at these positions by using the following primersdescribed in Park H-S et al., Science 311:535-538 (2006):

67XF, (SEQ ID NO: 19) 5′-GT ATC ACC ATC AAC ACT TCT NNK GTT GAA TAC GACACC CCG-3′; H67R, (SEQ ID NO: 20) 5′-AGA AGT GTT GAT GGT GAT AC-3′;216XF, (SEQ ID NO: 21)5′-CCG TTC CTG CTG CCG ATC NNK NNK GTA NNK TCC ATC TCC GGT CGT GGT-3′;216R, (SEQ ID NO: 22) 5′-GAT CGG CAG CAG GAA CGG-3′; 229XF,(SEQ ID NO: 23) 5′-GGT CGT GGT ACC GTT GTT NNK GGT CGT GTA GAA CGCGG-3′; 229R, (SEQ ID NO: 24) 5′-AAC AAC GGT ACC ACG ACC-3′; 274XF,(SEQ ID NO: 25) 5′-GAA GGC CGT GCT GGT GAG NNK GTA GGT GTT CTG CTGCG-3′; and 274R, (SEQ ID NO: 26) 5′-CTC ACC AGC ACG GCC TTC-3′.

The final PCR products were purified and digested with BamHI and SalI,and ligated into pKD-SepRS to generate the EF-Tu library. The ligatedvectors were transformed into E. coli Top10ΔserB containing pCAT112-SepTto generate a library of 3×10⁸ mutants. The unbiased mutation of thelibrary was confirmed by selecting twenty random clones and sequencingeach mutant tufB insert.

The mutant EF-Tu library was subjected to a first round of selection, inwhich clones sup-pressing the amber stop codon in the CAT gene cansurvive on LB plates supplemented with 10 mg/ml tetracycline (Tc), 25mg/ml Kan, 50 mg/ml chloramphenicol (Cm), 2 mM Sep, and 0.05 mMisopropyl-β-D-thiogalactopyranoside (IPTG). After 48 h incubation at30°C., a pool of 10⁴ colonies was collected from the plates for plasmidpreparation. The pKD-SepRS-EFTu plasmids were separated from thereporter plasmid by agarose gel electrophosis and isolated using theQiagen gel purification kit.

There is a possibility that mutations in the amino acid binding site ofEF-Tu could induce incorporation of natural amino acids in response tothe amber codon in the CAT gene, resulting in false positive clones. Toselect against these EF-Tu mutants, the pKD-SepRS-EFTu plasmids from thefirst positive selection were transformed into E. coli Top10ΔserBharboring pCcdB. The cells were plated onto LB agar supplemented with 25mg/ml Kan, 25 mg/ml Cm, and 0.1 mM IPTG. After 48 h incubation at 30°C.,twenty individual clones were picked and subjected to plasmidpurification to isolate pKD-SepRS-EFTu as described above. The EF-Tumutant genes were digested from the plasmid and recloned into pKD-SepRS.

Resulting pKD-SepRS-EFTu plasmids were transformed into E. coliTop10ΔserB containing pCAT112-SepT for a third round of selection whichwas carried out under the same conditions as the first. This time,individual colonies were isolated from agar plates and clones weretested for their ability to grow on Cm over a concentration range from 5to 100 mg/ml. Total plasmid was purified from isolates showing strong Cmresistance, and pKD-SepRS-EFTu plasmids were subjected to sequencing.

To confirm that the observed Cm resistance is dependent on the presenceof both, mutant EF-Tu and SepRS, EF-Tu mutant genes were excised fromtheir plasmids, recloned into pKD, and retransformed into E. coliTop10ΔserB containing pCAT112-SepT. Cells were then tested for Cmresistance as described above.

Expression and Purification of M. maripaludis SepRS and CysRS

SepRS and CysRS were produced in E. coli and purified as described byHohn, M. J. et al., Proc Natl Acad Sci USA 103, 18095 (2006).

Expression and Purification of EF-Tu and EF-Sep

pMAL-EFTu or pMAL-EFSep were transformed into E. coli BL21 (DE3) codonplus (Stratagene). A pre-culture was used to inoculate 1000 ml of LBbroth with 100 μg/ml of Amp, 34 μg/ml Cm, 5052 solution, and phosphatebuffer for autoinduction as described by Studier, F W. Protein ExprPurif 41, 207 (2005). The cells were grown for 6 h at 37°C. andcontinued at 20°C. for 18 h.

The cells were pelleted and lysed by shaking for 20 min. in BugBuster(Novagen) reagent supplemented with 50 mM Tris-HCl (pH 7.6), 60 mMNH₄Cl, 7 mM MgCl₂, 14.3 mM 2-mercapto-ethanol, 50 μM GDP, 10% glycerol,25 U ml⁻¹ Benzoase, 1 mg ml⁻¹ lysozyme, and Protease inhibitor cocktail(Roche).

The extract was clarified by ultracentrifugation and applied to aNi²⁺-NTA resin (Qiagen) and purified according to the manufacturer'sinstructions.

The eluted enzymes were dialyzed into 20 mM Hepes-KOH (pH 7.0). 40 mMKCl, 1 mM MgCl₂, 5 mM DTT, 50 μM GDP, and 30% glycerol. SDS-PAGEelectrophoresis followed by staining with Coomassie blue revealedgreater than 95% purity.

Results

Guided by the structure of the E. coli EF-Tu:Phe-tRNAPhe complex (P.Nissen et al., Science 270, 1464 (1995)) it was decided to randomizecertain positions in the amino acid binding pocket to evolve EF-Tuvariants that bind Sep-tRNA and promote its delivery to the ribosome.Six residues (His67, Asp216, Glu217, Phe219, Thr229, and Asn274) wereselected for complete randomization generating a library of 3×10⁸ EF-Tumutants. To select in vivo variants that permits Sep incorporation inthe presence of SepRS and tRNA^(Sep) three rounds of selections(positive, negative, positive) were performed that yielded severalclones with the desired phenotype. One clone, designated EF-Sep, wastested further in detail. While the combination of SepRS and EF-Sep wasnot active in the CAT suppression assay (FIG. 2, lane G), the furtherinclusion of tRNA^(Sep) led to a 10-fold increase in Cm resistance (FIG.2, lane H). Thus, it appeared that EF-Sep could bind Sep-tRNA^(Sep), afact that was ascertained in the hydrolysis protection assay (FIG. 3).The DNA sequence of the EF-Sep gene revealed the nature of themutations.

EF-Tu mutants (EF-Sep) that could bind Sep-tRNA include those having thefollowing amino acid sequences;

(EFSep-M6, SEQ ID NO: 1)MSKEKFERTKPHVNVGIGHVDHGKTTLTAAITTVLAKTYGGTARAFDQID NAPEEKARGITINTS RVEYDTPTRHYAHVDCPGHADYVKNMITGAAQMDGAILVVAATDGPMPQTREHILLGRQVGVPYIIVFLNKCDMVDDEELLELVEMEVRELLSQYDFPGDDTPIVRGSALKALEGDAEWEAKILILAGFLDSYIP EPERAIDKPFLLPI TR V YSISGRGTVV S GRVERGIIKVGEEVIEVGIKET QKSTCTGVEMFRKLLDEGRAGE FVGVLLRGIKREEIERGQVLAKPGTIKPHTKFESEVYILSKDEGGRHTPFFKGYRPQFYFRTTDVTGTIELPEGVEMVMPGDNIKMVVTLIHPIAMDDGLRFAIREGGRTVGAGVVAKVLRDPNSSSV DKLAAALE(EFSep-M7, SEQ ID NO: 2)MSKEKFERTKPHVNVGTIGHVDHGKTTLTAAITTVLAKTYGGAARAFDQI DNAPEEKARGITINTS RVEYDTPTRHYAHVDCPGHADYVKNMITGAAQMDGAILVVAATDGPMPQTREHILLGRQVGVPYIIVFLNKCDMVDDEELLELVEMEVRELLSQYDFPGDDTPIVRGSALKALEGDAEWEAKILELAGFLDSYI PEPERAIDKPFLLPI TY VY SISGRGTVV S GRVERGIIKVGEEVEIVGI N E TQKSTCTGVEMFRKLLDEGRAGE AVGVLLRGIKREEIERGQVLAKPGTIKPHTKFESEVYILSKDEGGRHTPFFKGYRPQFYFRTTDVTGTIELPEGVEMVMPGDNIKMVVTLIHPIAMDDGLRFAIREGGRTVGAGVVAKVLRDPNSSS VDKLAAALE(EFSep-M8, SEQ ID NO: 3)MSKEKFERTKPHVNVGTIGHVDHGKTTLTAAITTVLAKTYGGAARAFDQI DNAPEEKARGITINTS RVEYDTPTRHYAHVDCPGHADYVKNMITGAAQMDGAILVVAATDGPMPQTREHILLGRQVGVPYIIVFLNKCDMVDDEELLELVEMEVRELLSQYDFPGDDTPIVRGSALKALEGDAEWEAKILELAGFLDSYI PEPERAIDKPFLLPI NG VY SISGRGTVV S GRVERGIIKVGEEVEIVGIKE TQKSTCTGVEMFRKLLDEGRAGE WVGVLLRGIKREEIERGQVLAKPGTIKPHTKFESEVYILSKDEGGRHTPFFKGYRPQFYFRTTDVTGTIELPEGVEMVMPGDNIKMVVTLIHPIAMDDGLRFAIREGGRTVGAGVVAKVLRDPNSSS VDKLAAALE(EFSep-M9, SEQ ID NO: 4)MSKEKFERTKPHVNVGTIGHVDHGKTTLTAAITTVLAKTYGGAARARFQI DNAPEEKARGITINTS RVEYDTPTRHYAHVDCPGHADYVKNMITGAAQMDGAILVVAATDGPMPQTREHILLGRQVGVPYIIVFLNKCDMVDDEELLELVEMEVRELLSQYDFPGDDTPIVRGSALKALEGDAEWEAKILELAGFLDSYI PEPERAIDKPFLLPI TA VY SISGRGTVV S GRVERGIIKVGEEVEIVGIKE TQKSTCTGVEMFRKLLDEGRAGE AVGVLLRGIKREEIERGQVLAKPGTIKPHTKFESEVYILSKDEGGRHTPFFKGYRPQFYFRTTDVTGTIELPEGVEMVMPGDNIKMVVTLIHPIAMDDGLRFAIREGGRTVGAGVVAKVLRDPNSSS VDKLAAALE

Nucleic acid encoding EF-Tu mutants (EFSep) that could bind Sep-tRNAinclude those having the following amino acid sequences:

(EFSep-M6, SEQ ID NO: 5)ATGTCTAAAGAAAAGTTTGAACGTACAAAACCGCACGTTAACGTCGGTACTATCGGCCACGTTGACCATGGTAAAACAACGCTGACCGCTGCAATCACTACCGTACTGGCTAAAACCTACGGCGGT A CTGCTCGCGCATTCGACCAGATCGATAACGCGCCGGAAGAAAAAGCTCGTGGTATCACCATCAACACTTCT CG GGTTGAATACGACACCCCGACCCGTCACTACGCACACGTAGACTGCCCGGGGCACGCCGACTATGTTAAAAACATGATCACCGGTGCTGCGCAGATGGACGGCGCGATCCTGGTAGTTGCTGCGACTGACGGCCCGATGCCGCAGACTCGTGAGCACATCCTGCTGGGTCGTCAGGTAGGCGTTCCGTACATCATCGTGTTCCTGAACAAATGCGACATGGTTGATGACGAAGAGCTGCTGGAACTGGTTGAAATGGAAGTTCGTGAACTTCTGTCTCAGTACGACTTCCCGGGCGACGACACTCCGATCGTTCGTGGTTCTGCTCTGAAAGCGCTGGAAGGCGACGCAGAGTGGGAAGCGAAAATCCTGGAACTGGCTGGCTTCCTGGATTCTTACATTCCGGAACCAGAGCGTGCGATTGACAAGCCGTTCCTGCTGCCGATC ACCCG G GTAT ACTCCATCTCCGGTCGTGGTACCGTTGTT T C G GGTCGTGTAGAACGCGGTATCATCAAAGTTGGTGAAGAAGTTGAAATCGTTGGTATCAAAGAGACTCAGAAGTCTACCTGTACTGGCGTTGAAATGTTCCGCAAACTGCTGGA CGAAGGCGTGCTGGTGAG TTCGTAGGTGTTCTGCTGCGTGGTATCAAACGTGAAGAAATCGAACGTGGTCAGGTACTGGCTAAGCCGGGCACCATCAAGCCGCACACCAAGTTCGAATCTGAAGTGTACATTCTGTCCAAAGATGAAGGCGGCCGTCATACTCCGTTCTTCAAAGGCTACCGTCCGCAGTTCTACTTCCGTACTACTGACGTGACTGGTACCATCGAACTGCCGGAAGGCGTAGAGATGGTAATGCCGGGCGACAACATCAAAATGGTTGTTACCCTGATCCACCCGATCGCGATGGACGACGGTCTGCGTTTCGCAATCCGTGAAGGCGGCCGTACCGTTGGCGCGGGCGTTGTAGCAAAAGTTCTGAGGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAG (EFSep-M7, SEQ ID NO: 6)ATGTCTAAAGAAAAGTTTGAACGTACAAAACCGCACGTTAACGTCGGTACTATCGGCCACGTTGACCATGGTAAAACAACGCTGACCGCTGCAATCACTACCGTACTGGCTAAAACCTACGGCGGTGCTGCTCGCGCATTCGACCAGATCGATAACGCGCCGGAAGAAAAAGCTCGTGGTATCACCATCAACACTTCT AG GGTTGAATACGACACCCCGACCCGTCACTACGCACACGTAGACTGCCCGGGGCACGCCGACTATGTTAAAAACATGATCACCGGTGCTGCGCAGATGGACGGCGCGATCCTGGTAGTTGCTGCGACTGACGGCCCGATGCCGCAGACTCGTGAGCACATCCTGCTGGGTCGTCAGGTAGGCGTTCCGTACATCATCGTGTTCCTGAACAAATGCGACATGGTTGATGACGAAGAGCTGCTGGAACTGGTTGAAATGGAAGTTCGTGAACTTCTGTCTCAGTACGACTTCCCGGGCGACGACACTCCGATCGTTCGTGGTTCTGCTCTGAAAGCGCTGGAAGGCGACGCAGAGTGGGAAGCGAAAATCCTGGAACTGGCTGGCTTCCTGGATTCTTACATTCCGGAACCAGAGCGTGCGATTGACAAGCCGTTCCTGCTGCCGATC ACCT A CGTAT ACTCCATCTCCGGTCGTGGTACCGTTGTT T C G GGTCGTGTAGAACGCGGTATCATCAAAGTTGGTGAAGAAGTTGAAATCGTTGGTATCAA T GAGACTCAGAAGTCTACCTGTACTGGCGTTGAAATGTTCCGCAAACTGCTGGA CGAAGGCCGTGCTGGTGAGGCG GTAGGTGTTCTGCTGCGTGGTATCAAACGTGAAGAAATCGAACGTGGTCAGGTACTGGCTAAGCCGGGCACCATCAAGCCGCACACCAAGTTCGAATCTGAAGTGTACATTCTGTCCAAAGATGAAGGCGGCCGTCATACTCCGTTCTTCAAAGGCTACCGTCCGCAGTTCTACTTCCGTACTACTGACGTGACTGGTACCATCGAACTGCCGGAAGGCGTAGAGATGGTAATGCCGGGCGACAACATCAAAATGGTTGTTACCCTGATCCACCCGATCGCGATGGACGACGGTCTGCGTTTCGCAATCCGTGAAGGCGGCCGTACCGTTGGCGCGGGCGTTGTAGCAAAAGTTCTGAGGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAG (EFSep-M8, SEQ ID NO: 7)ATGTCTAAAGAAAAGTTTGAACGTACAAAACCGCACGTTAACGTCGGTACTATCGGCCACGTTGACCATGGTAAAACAACGCTGACCGCTGCAATCACTACCGTACTGGCTAAAACCTACGGCGGTGCTGCTCGCGCATTCGACCAGATCGATAACGCGCCGGAAGAAAAAGCTCGTGGTATCACCATCAACACTTCT CG GGTTGAATACGACACCCCGACCCGTCACTACGCACACGTAGACTGCCCGGGGCACGCCGACTATGTTAAAAACATGATCACCGGTGCTGCGCAGATGGACGGCGCGATCCTGGTAGTTGCTGCGACTGACGGCCCGATGCCGCAGACTCGTGAGCACATCCTGCTGGGTCGTCAGGTAGGCGTTCCGTACATCATCGTGTTCCTGAACAAATGCGACATGGTTGATGACGAAGAGCTGCTGGAACTGGTTGAAATGGAAGTTCGTGAACTTCTGTCTCAGTACGACTTCCCGGGCGACGACACTCCGATCGTTCGTGGTTCTGCTCTGAAAGCGCTGGAAGGCGACGCAGAGTGGGAAGCGAAAATCCTGGAACTGGCTGGCTTCCTGGATTCTTACATTCCGGAACCAGAGCGTGCGATTGACAAGCCGTTCCTGCTGCCGATC A A C GG GG TAT ACTCCATCTCCGGTCGTGGTACCGTTGTT T C G GGTCGTGTAGAACGCGGTATCATCAAAGTTGGTGAAGAAGTTGAAATCGTTGGTATCAAAGAGACTCAGAAGTCTACCTGTACTGGCGTTGAAATGTTCCGCAAACTGCTGGA CGAAGGCCGTGCTGGTGAGTGG GTAGGTGTTCTGCTGCGTGGTATCAAACGTGAAGAAATCGAACGTGGTCAGGTACTGGCTAAGCCGGGCACCATCAAGCCGCACACCAAGTTCGAATCTGAAGTGTACATTCTGTCCAAAGATGAAGGCGGCCGTCATACTCCGTTCTTCAAAGGCTACCGTCCGCAGTTCTACTTCCGTACTACTGACGTGACTGGTACCATCGAACTGCCGGAAGGCGTAGAGATGGTAATGCCGGGCGACAACATCAAAATGGTTGTTACCCTGATCCACCCGATCGCGATGGACGACGGTCTGCGTTTCGCAATCCGTGAAGGCGGCCGTACCGTTGGCGCGGGCGTTGTAGCAAAAGTTCTGAGGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAG (EFSep-M9, SEQ ID NO: 8)ATGTCTAAAGAAAAGTTTGAACGTACAAAACCGCACGTTAACGTCGGTACTATCGGCCACGTTGACCATGGTAAAACAACGCTGACCGCTGCAATCACTACCGTACTGGCTAAAACCTACGGCGGTGCTGCTCGCGCATTCGACCAGATCGATAACGCGCCGGAAGAAAAAGCTCGTGGTATCACCATCAACACTTCTC G GGTTGAATACGACACCCCGACCCGTCACTACGCACACGTAGACTGCCCGGGGCACGCCGACTATGTTAAAAACATGATCACCGGTGCTGCGCAGATGGACGGCGCGATCCTGGTAGTTGCTGCGACTGACGGCCCGATGCCGCAGACTCGTGAGCACATCCTGCTGGGTCGTCAGGTAGGCGTTCCGTACATCATCGTGTTCCTGAACAAATGCGACATGGTTGATGACGAAGAGCTGCTGGAACTGGTTGAAATGGAAGTTCGTGAACTTCTGTCTCAGTACGACTTCCCGGGCGACGACACTCCGATCGTTCGTGGTTCTGCTCTGAAAGCGCTGGAAGGCGACGCAGAGTGGGAAGCGAAAATCCTGGAACTGGCTGGCTTCCTGGATTCTTACATTCCGGAACCAGAGCGTGCGATTGACAAGCCGTTCCTGCTGCCGATC ACC G C G GTAT ACTCCATCTCCGGTCGTGGTACCGTTGTTTCGGGTCGTGTAGAACGCGGTATCATCAAAGTTGGTGAAGAAGTTGAAATCGTTGGTATCAAAGAGACTCAGAAGTCTACCTGTACTGGCGTTGAAATGTTCCGCAAACTGCTGGA CGAAGGCCGTGCTGGTGAGGC CGTAGGTGTTCTGCTGCGTGGTATCAAACGTGAAGAAATCGAACGTGGTCAGGTACTGGCTAAGCCGGGCACCATCAAGCCGCACACCAAGTTCGAATCTGAAGTGTACATTCTGTCCAAAGATGAAGGCGGCCGTCATACTCCGTTCTTCAAAGGCTACCGTCCGCAGTTCTACTTCCGTACTACTGACGTGACTGGTACCATCGAACTGCCGGAAGGCGTAGAGATGGTAATGCCGGGCGACAACATCAAAATGGTTGTTACCCTGATCCACCCGATCGCGATGGACGACGGTCTGCGTTTCGCAATCCGTGAAGGCGGCCGTACCGTTGGCGCGGGCGTTGTAGCAAAAGTTCTGAGGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAG

Example 3: Demonstration of Sep Incorporation into Myoglobin Materialsand Methods Construction of Plasmids

pMYO127TAG-SepT was constructed by cloning a codon-optimized andC-terminally His₆-tagged sperm whale myoglobin gene under the control ofthe lpp promoter between NotI and BglII in pSepT. An amber stop codonwas introduced to the myoglobin gene at position Asp127 by quickchangemutagenesis. The nucleotide sequence of the codon-optimized myoglobingene is as follows:

(SEQ ID NO: 44) ATGGTTCTGTCTGAAGGTGAATGGCAGCTGGTTCTGCACGTTTGGGCTAAAGTTGAAGCTGACGTTGCTGGTCACGGTCAGGACATCCTGATCCGTCTGTTCAAATCTCACCCGGAAACCCTGGAAAAATTCGACCGTTTCAAACACCTGAAAACCGAAGCTGAAATGAAGGCTTCTGAAGACCTGAAAAAACACGGTGTTACCGTTCTGACCGCTCTGGGTGCTATCCTGAAGAAAAAGGGTCACCACGAAGCTGAACTGAAACCGCTGGCTCAGTCTCACGCTACCAAACACAAAATCCCGATCAAATACCTGGAGTTCATCTCTGAAGCTATCATCCACGTTCTGCACTCTCGTCATCCGGGTAACTTCGGTGCTGACGCTCAGGGTGCTATGAACAAAGCTCTGGAACTGTTCCGTAAAGACATCGCTGCTAAATACAAAGAACTGGGTTACCAGGGTGGTTCTGGTCATCACCATCACCATCACTAA.

Results

To prove that the observed suppression is due to Sep incorporation amyoglobin variant with an amber codon in position 127 (normally Asp) anda C-terminal His₆-tag was expressed. The expected full length proteinwas synthesized (yield is 2 mg/L of culture) only when EF-Sep, SepRS andtRNA^(Sep) were co-expressed. The amino acid incorporated via EF-Sep inresponse to the amber codon was identified by analyzing both the intactand trypsin-digested Myo-His₆ mutant protein. MS-TOF and MS/MS analysisshow that Sep is present at the position specified by UAG.

Example 4: Active MEK Synthesis In Vivo Materials and MethodsConstruction of Plasmids

pET15-ERK2 encodes N-terminally His₆-tagged mitogen-activated proteinkinase (Erk2) under the control of a T7 promoter. The human Erk2 genewas PCR amplified from plasmid BC017832 (ATCC) using primers ERK2-F(5′-GGA ATT CCA TAT GGC GGC GGC GGC GGC G-3′, SEQ ID NO:27) and ERK2-R(5′-CCG CTC GAG TTA AGA TCT GTA TCC TGG-3′, SEQ ID NO:28). The PCRproduct was cloned between NdeI and XhoI in vector pET15b (Novagen).

pET20-MBPMEK1 encodes a fusion protein consisting of human MEK1 with anN-terminal maltose binding protein (MBP) tag and a C-terminal His₆-tag.The gene encoding human MEK1 which was codon-optimized for E. coli andcustom-synthesized in vitro (Genscript), was cloned between EcoRI andPstI into pMALc2x (New England Biolabs). The resulting MBP-MEK1 fusionconstruct was then amplified with primers ET20MEKF (5′-AAG GAA ATT AATGAA AAT CGA AGA AGG TAA-3′, SEQ ID NO:29) and ET20MEKR (5′-CTA GAG GATCCG GCG CGC-3′, SEQ ID NO:30) adding AseI and BamHI restriction sites,and the PCR product was ligated between NdeI and BamHI into pET20b.

Nucleotide Sequence of Codon-optimized MEK1

(SEQ ID NO: 31) ATGCCGAAGAAGAAACCGACCCCGATCCAGCTGAACCCGGCTCCGGACGGTTCTGCTGGTTAACGGCACCTCTTCTGCTGAAACCAACCTGGAAGCTCTGCAAAAGAAACTGGAAGAACTGGAACTGGACGAACAGCAGCGTAAACGTCTGGAAGCGTTCCTGACCCAGAAACAGAAAGTTGGTGAACTGAAAGACGACGACTTCGAAAAAATCTCTGAACTGGGTGCTGGTAACGGTGGTGTTGTTTTCAAAGTTTCTCACAAACCGTCCGGTCTGGTTATGGCTCGTAAACTGATCCACCTGGAAATCAAACCGGCTATCCGTAACCAGATCATCCGTGAACTGCAAGTTCTGCACGAATGCAACTCTCCGTACATCGTTGGTTTCTACGGTGCTTTCTACTCTGACGGTGAAATCTCTATCTGCATGGAACACATGGACGGTGGTTCTCTGGACCAGGTTCTGAAAAAAGCTGGTCGTATCCCGGAACAGATCCTGGGTAAAGTTTCTATCGCTGTTATCAAAGGTCTGACCTACCTGCGTGAAAAACACAAAATCATGCACCGTGACGTTAAACCGTCTAACATCCTGGTTAACTCTCGTGGTGAAATCAAACTGTGCGACTTCGGTGTTTCTGGTCAGCTGATCGACTCTATGGCTAACTCTTTCGTTGGCACCCGTTCTTACATGTCTCCGGAACGTCTGCAAGGCACCCACTACTCTGTTCAGTCTGACATCTGGTCTATGGGTCTGTCTCTGGTTGAAATGGCTGTTGGTCGTTACCCGATCCCGCCGCCGGACGCTAAAGAACTGGAACTGATGTTCGGTTGCCAGGTTGAAGGTGACGCTGCTGAAACCCCGCCGCGTCCGCGTACTCCGGGTCGTCCGCTGTCTTCTTACGGTATGGACTCTCGTCCGCCGATGGCTATCTTCGAACTGCTGGACTACATCGTTAACGAACCGCCGCCGAAACTGCCGTCTGGTGTTTTCTCTCTGGAGTTCCAGGACTTCGTTAACAAATGCCTGATCAAAAACCCGGCTGAACGTGCTGACCTGAAACAGCTGATGGTTCACGCTTTCATCAAACGTTCTGACGCTGAAGAAGTTGACTTCGCTGGTTGGCTGTGCTCTACCATCGGTCTGAACCAGCCGTCTACCCCGACCCACGCTGCTGGTGTGGCAGCCGCAGCTGCGCATCA TCACCACCATCACTAA.

pCG-MBPMEK1SS was generated by the ligation of three PCR products. OnePCR product was derived from pGFIB (Normanly J, et al. Nature 321:213(1986)) using primers GFIB-F (5′-ATA AGA ATG CGG CCG CGC CGC AGC CGA ACGACC GAG-3′, SEQ ID NO:32) and GFIB-R (5′-CTA GCT AGC GTC TGA CGC TCA GTGGAA CG-3′, SEQ ID NO:33). The second PCR product was generated frompCDFDuet-1 (Novagen) using primers CDF-F (5′-CTA GCT AGC TCA CTC GGT CGCTAC GCT-3′, SEQ ID NO:34) and CDF-R (5′-ATA AGA ATG CGG CCG CTG AAA TCTAGA GCG GTT CAG-3′, SEQ ID NO:35). Both PCR products were digested withNheI and NotI and ligated to form plasmid pCG, The third PCR product,encoding an expression cassette for MBP-MEK1-His₆ under the control ofT7 promoter and T7 terminator, was generated from pET20-MBPMEK1 usingprimers ETCDGFF (5′-AAA AGG CGC CGC CAG CCT AGC CGG GTC CTC AAC G-3′,SEQ ID NO:36) and ETCDGFR (5′- AAC TGC AGC CAA TCC GGA TAT AGT TC-3′,SEQ ID NO:37). This PCR product was cloned between the NarI and PstIsites of pCG.

The codon for Ser 222 in MEK1 was then replaced by a GAA codon (encodingGlu) using Quickchange mutagenesis (Stratagene). In the same way, codonSer 218 was either changed to GAA to generate pCG-MBPMEK1EE, or to anamber stop codon, resulting in pCG-MBPMEK1XE. In pCG-MBPMEK1XS only thecodon for Ser218 was changed to UAG and in pCG-MBPMEK1XX both codons forSer218 and Ser222 were changed to amber.

Expression and Purification of Myoglobin

To express mutant myoglobin, pKD-SepRS-EF-Sep and pKD-SepRS weretransformed into E. coli Top10ΔserB containing pMYO127TAG-SepT. E. coliTop10ΔserB with pMYO, encoding the wild type myoglobin gene was used asa control. Cultures were grown in LB medium supplemented with 2 mM Sep.When A₆₀₀ reached 0.6 protein expression was induced with 0.05 mM IPTGfor 12 h at 25°C. The cells were harvested, resuspended in lysis buffer(50 mM Tris-HCl (pH 7.8), 300 mM NaCl, 14.3 mM 2-mercaptoethanol)supplemented with protease inhibitor cocktail (Roche), and subjected tosonication. The lysate was centrifuged at 10,000×g for 30 min and thesupernatant was applied to Ni²⁺-NTA agarose (Qiagen) purificationaccording to the manufacturer's instruction.

Expression and Purification of MEK1

To express MEK1 (as a maltose binding protein fusion-protein) E. coliBL21ΔserB was transformed with plasmids pKD-SepRS-EFSep.pCAT112TAG-SepT, and pCG-MBPMEK1SS, pCG-MBPMEK1EE, pCG-MBPMEK1XE,pCG-MBPMEK1XS, or pCG-MBPMEK1XX, respectively. Plasmid pCAT112TAG-SepTwas replaced by pL11C-SepT in the strain used to produceMBP-MEK1(Sep218,Ser222)-His₆ for mass spectrometry analysis.

Cells were grown at 30° C. in 1 liter of LB supplemented with 100 μg/mlof Amp, 50 μg/ml Kan, 12 μg/ml Tc, 2 mM Sep, 5052 solution, andphosphate buffer for autoinduction. When A600 reached 0.6, temperaturewas changed to 16° C. and incubation continued for 18 h. Afterharvesting, cells were lysed in 20 ml BugBuster reagent containing 50 mMTris-HCl (pH 7.8), 500 mM NaCl, 0.5 mM EGTA. 0.5 mM EDTA, 14.3 mM2-mercapto-ethanol, 10% glycerol, 0.03% Brij-35, protease inhibitors, 25U ml⁻¹ Benzoase, and 1 mg ml⁻¹ lysozyme. The lysate was clarified byultracentrifugation, and applied to a 0.4 ml Ni²⁺-NTA agarose column.The column was washed with 15 ml wash buffer (50 mM Tris-HCl (pH 7.8),150 mM NaCl, 0.5 mM EGTA, 0.5 mM EDTA, 14.3 mM 2-mercaptoethanol, 10%glycerol, 0.03% Brij-35, and 20 mM imidazole). Proteins were eluted in0.8 ml of wash buffer supplemented with 300 mM imidazole, dialyzedagainst 50 mM Tris-HCl (pH 7.8), 150 mM NaCl, 0.1 mM EGTA, 5 mM DTT, 30%glycerol, and 0.03% Brij-35, and stored at −20° C. Purified proteinswere analyzed by SDS-PAGE.

Expression and Purification of Erk2

E. coli BL21 (DE3) codon plus cells were transformed with pET15-ERK2 andgrown at 37° C. in 1 liter LB broth supplemented with 100 g/ml Amp and34 g/ml Cm. When the cultures reached A600 of 0.6, 0.2 mM IPTG was addedand expression was induced for 19 h at 16° C.

Cell lysis, Ni²⁺purification, and dialysis of Erk2 were carried out asdescribed for MEK1. Erk2 was 99% pure, as judged by Coomassie brilliantblue staining after SDS-PAGE.

Preparation and Aminoacylation of tRNA.

Total tRNA from E. coli Top10 or from E. coli Top10 complemented withpCysT or pSepT, respectively, was purified by standard procedures andacylated with [¹⁴C]Sep by M. maripaludis SepRS as described previously.In vivo synthesized tRNA was for this experiment to ensure thatnucleoside modifications introduced into tRNA by E. coli modifyingenzymes do not affect tRNA recognition by SepRS. M. jannaschiitRNA^(Cys) contains m¹G37 when isolated from M. jannaschii. Since the E.coli methylase TrmD is known to methylate G537 of archaeal tRNA^(Pro),it is believed that the in vivo expressed tRNA^(Sep) also carries them¹G37 modification. In vitro transcript of M. jannaschii tRNA^(Cys) wasprepared and acylated with [¹⁴C]Sep or [³⁵S]Cys using recombinant M.maripaludis SepRS or CysRS. M. jannaschii tRNA^(Cys) transcript waschosen for these experiments because of the poor folding properties ofin vitro transcribed M. maripaludis tRNA^(Cys) (Hohn, M. J. Proc NatlAcad Sci USA 103, 18095 (2006)).

EF-Tu Hydrolysis Protection Assays

To assay hydrolysis protection of acylated tRNA^(Cys) by EF-Tu, MmptRNA^(Cys) in vitro transcripts acylated with [¹⁴C]Sep or [³⁵S]Cys,respectively, were phenol/chlorophorm extracted, and the aqueous phasewas passed over Sephadex® G25 Microspin columns (GE Healthcare)equilibrated with water. Protection of aminoacylated tRNA by EF-Tu wasassayed as described earlier with slight modifications (Ling J. et al.,Proc Natl Acad Sci USA 104, 15299 (2007)). Briefly, EF-Tu or EF-Sep(both purified as maltose binding protein fusion proteins) wereactivated for 20 min. at 37° C. in buffer containing 100 mM Tris-HCl (pH8.2), 120 mM NH4Cl, 7 mM MgCl₂, 5 mM DTT, 5 mM phosphoenolpyruvate, 1.5mM GTP, and 0.12 μg/μl pyruvate kinase. Hydrolysis of 2 μM[¹⁴C]Sep-tRNA^(Cys) was then monitored at 25° C. in the presence of 40μM EF-Tu (wt), EF-Sep, or BSA, respectively. Aliquots were taken fromthe reaction mix at indicated time points and spotted on 3 MM filterdiscs presoaked with 10% trichloroacetic acid. Filters were washed with5% trichloroacetic acid, dried, and radioactivity was measured by liquidscintillation counting.

MEK Activity Assays

Recombinant MEK1 variants were assayed (as maltose binding protein (MBP)fusion-proteins). Briefly, in a first reaction, various amounts(2.5-5000 ng) of recombinant MBP-MEK1 variants were used tophosphorylate (and activate) bacterially expressed MAP kinase (Erk2) for15 min. at 30° C. in 35 μl kinase assay buffer containing 12 mM MOPS pH7.2, 20 mM MgCl₂, 3 mM EGTA, 15 mM β-glycerol phosphate, 0.6 mM DTT, 140μM ATP, and 1 μg Erk2.

After 15 min, a 5 μl aliquot was transferred to a second reaction inwhich activated Erk2 phosphorylates myelin basic protein (MBP: 570 μgml¹) in kinase assay buffer in the presence of [γ-³²P]ATP. After 15 min.incubation at 30° C. 25 μl aliquots were transferred onto p81phospho-cellulose filters (Whatman). The filters were washed three timeswith 180 mM phosphoric acid and then rinsed with acetone.Phosphorylation was quantitated by scintillation counting and thespecific activity of MEK1 was calculated from the amount of[³²P]phosphate incorporated into MBP.

LC and MS/MS Conditions for Multiple Reaction Monitoring (MRM)

Purified MEK1 proteins were separated by SDS-PAGE, visualized withComassie stain, excised, washed in 50% acetonitrile (ACN)/50 mM NH₄HCO₃,crushed, and digested at 37° C. in a 20 μg/ml trypsin (Promega) solutionin 10 mM NH₄HCO₃. Digested peptides in solution were dried and dissolvedin 3 μl of 70% formic acid (FA), and then diluted to 10 μl with 0.1%TFA. Peptides for MRM were synthesized at the KECK peptide synthesisfacility at Yale. The human MEK peptide LCDFGVSGQLIDS*MANSFVGTR (SEQ IDNO:40) (*phospho-Ser; YPED peptide ID, SOL14075) was synthesized topermit the development of a specific method for quantitative MRM. Crudesynthetic peptides were direct infused at a concentration of ˜10 pmol/μland Collision Energy and Declustering Potentials of the transitions wereoptimized. LC-MRM was performed on an ABI 5500 QTRAP triple quadruplemass spectrometer inter-faced with a Waters nanoAcquity UPLC systemrunning Analyst 1.5 software. Peptides were resolved for MRM (LC step)by loading 4 μl of sample onto a Symmetry C18 nanoAcquity trappingcolumn (180 μm×20 mm 5 μm) with 100% water at 15 μl per minute for 1minute. After trapping, peptides were resolved on a BEH130 C18nanoAcquity column (75 μm×50 mm 1.7 μm) with a 30 minute. 2-40%*ACN/0.1% FA linear gradient. (0.5 μl/min flow rate). MRM scanning wascarried out with 18 transitions and a cycle time of 1.44 seconds with a40 millisecond dwell time per transition. An MRM Initiated Detection andSequencing (MIDAS) was performed. The IDA method consisted of the mostintense peak using rolling collision energy. The target ions wereexcluded after 3 occurrences for 30 seconds. The EPI scan had a scanrate of 20,000 Da/sec with a sum of 3 scans and mass range of 100-1000Da and a cycle time of 1.4 msec. Files were searched using Mascotversion 2.3 with the Swissprot database (08/2010) selected (humantaxonomic restriction,). Phosphorylated S and T, and propionamide C werevariable modifications. Peptide and fragment mass tolerance is 0.6 Da,with 1 missed cleavage. Quantification was performed using MultiQuant2.0.

Results

To further demonstrate the usefulness of the disclosed strategy for thesynthesis of a protein that is naturally phosphorylated at a serineresidue, recombinant, Sep containing mitogen-activated ERK activatingkinase 1 (MEK1) was produced. This key enzyme of the mitogen-activatedsignaling cascade in eukaryotic cells plays crucial roles in cellproliferation, cell development and differentiation, cell cycle controland oncogenesis (Sebolt-Leopold, J. S., et al. Nat Rev Cancer 4, 937(2004)). Activation of MEK1 requires post-translational phosphorylationof Ser218 and Ser222 by MEK activating kinases (e.g., Raf-1, MEKK, orMOS). Change of both Ser residues to Glu yields a constitutively activeenzyme albeit with lower activity (Alessi D R. et al., EMBO J 13, 1610(1994)).

To improve expression of this human protein in the E. coli BL21 ΔserBstrain and to allow purification by Ni²⁺-affinity chromatography a MEK1clone was designed to generate an N-terminal fusion with maltose bindingprotein (MBP) and with a C-terminal His₆-tag. Position 222 was changedto Glu and the codon for Ser2l8 was replaced by UAG to encode Sep. Afterexpression in the presence of SepRS, tRNA^(Sep) and EF-Sep 25 μg offull-length MBP-MEK1(Sep218,Glu222) were isolated from 1 L of culture.The presence of Sep in this recombinant MEK1-fusion protein wasdemonstrated by its activity in phosphorylating ERK2. The assay requiresthe additional component, myelin basic protein (MyBP) which will bephosphorylated by activated ERK2 in the presence of [γ-³²P]ATP; theamount of [³²P]MyBP relates to the specific activity of MEK1. As FIG. 4shows, MBP-MEK1 (Sep218,Glu222) had a 2,500-fold higher specificactivity than non-phosphorylated MBP-MEK1 (Ser218,Ser222), and a 70-foldhigher specific activity than the constitutively activeMBP-MEK1(Glu218,Glu222) mutant (FIG. 4).

To demonstrate the incorporation of Sep at position 218 an assay wasdeveloped utilizing multiple reaction monitoring (MRM) and atriple-quadrupole mass spectrometer. The MRM assay was designed todetect an intact tryptic phosphopeptide ion (m/z 823.4⁺³) derived fromMBP-MEK1(Sep218,Ser222) and 4 fragment ions produced bycollision-induced dissociation of this intact phosphopeptide (Table 1).The MRM method included an Information Dependent Acquisition (IDA) stepthat triggered a full MS/MS scan once the 823.4⁺³ ion, and associatedfragment ions, were detected. The IDA MS/MS spectrum confirmed theincorporation of Sep at position 218 and Ser at 222 in MBP-MEK1 (Sep218,Ser222).

TABLE 1 Peptide information for MRM precursor/ Peptide (SEQ ID NO: 40)product ion CE DP LC*DFGVSGQLIDS^(P)MANSFVGTR 823.4⁽

⁾/ 30.85 160.9 333.2⁽

⁾[y3] LC*DFGVSGQLIDS^(P)MANSFVGTR 823.4⁽

⁾/ 38.26 160.9 666.35⁽

⁾[y6] LC*DFGVSGQLIDS^(P)MANSFVGTR 823.4⁽

⁾/ 38.62 160.9 780.4⁽

⁾[y7] LC*DFGVSGQLIDS^(P)MANSFVGTR 823.4⁽

⁾/ 38.12 160.9 851.4⁽

⁾[y8] S^(P), phosphoserine, C*, propionamide; CE, Collision energy; DP,Dilution Potential

indicates data missing or illegible when filed

To determine if our E. coli expression system would allow thesimultaneous insertion of two Sep residues into the protein, the Sercodons in positions 218 and 222 were changed to UAG. As expected theexpression efficiency of MBP-MEK1 (Sep218,Sep222) was dramaticallyreduced compared to wild-type MBP-MEK1 (only about 1 μg of full lengthprotein was obtained from 1 L culture). The presence of Sep at bothactive site positions of MEK1 was tested by Western blot analysis usinga monoclonal antibody specific to the phosphorylated active site ofhuman MEK2. Only recombinant MBP-MEK1(Sep218,Sep222), and to a weakerextent MBP-MEK1(Sep218,Ser222) was detected in this experiment, whileneither MBP-MEK1(Ser218,Ser222), MBP-MEK(Sep218,Glu222) orMBP-MEK(Glu218,Glu222) was recognized by this antibody. The presence offull-length MBP-fusion proteins was confirmed by Coomassie staining andby Western hybridization with an MBP-specific antibody. Thisdemonstrates that the addition of SepRS, tRNA^(Sep) and EF-Sep endows E.coli with the ability to read UAG as a phosphoserine codon.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs.

1. A method of making a target protein, comprising expressing amessenger RNA (mRNA) encoding the target protein in a system comprising:an O-phosphoseryl-tRNA synthetase (SepRS) that preferentiallyaminoacylates a tRNA (tRNA^(Sep)) with phosphoserine; a tRNA^(Sep) thatcan be aminoacylated with phosphoserine by SepRS to form aSep-tRNA^(Sep) and recognize at least one codon in the mRNA encoding thetarget protein; and a mutant elongation factor (EF-Sep) that bindsSep-tRNA^(Sep); wherein the SepRS preferentially aminoacylates thetRNA^(Sep) with phosphoserine and the resulting Sep-tRNA^(Sep)recognizes at least one codon such that phosphoserine is incorporatedduring translation to form the target protein.
 2. The method of claim 1,wherein the EF-Sep comprises an amino acid sequence at least 90%identical to any one of SEQ ID NOS:1-4, wherein (i) the amino acidsequence comprises the amino acids of any one of SEQ ID NOS:1-4 atpositions corresponding to amino acid number 67, 216, 217, 219, 229, and274 of any one of SEQ ID NOS:1-4; or (ii) the amino acid sequencecomprises the amino acids of any one of SEQ ID NOS:1-4 at positionscorresponding to amino acid number 67, 217, 219, 229, and 274 of any oneof SEQ ID NOS:1-4 and a substitution mutation corresponding to aminoacid number 216 of any one of SEQ ID NOS:1-4.
 3. The method of claim 1,wherein the EF-Sep comprises an amino acid binding pocket foraminoacylated tRNA, wherein the binding pocket comprises (i) the bindingpocket for aminoacylated tRNA of any one of SEQ ID NOS: 1-4, or any oneof SEQ ID NO:1-4; or (ii) the binding pocket for aminoacylated tRNA ofSEQ ID NO:3 with a substitution at amino acid reside 216, optionallywherein the substitution is an asparagine-to-valine substitution(N216V).
 4. The method of claim 1, wherein the tRNA^(Sep) iscysteinyl-tRNA from Methanocaldococcus jannaschii.
 5. The method ofclaim 4, wherein the tRNA^(Sep) is encoded by a the nucleic acidsequence SEQ ID NO:41.
 6. The method of claim 1, wherein the SepRS isthe phosphoseryl-tRNA synthetase from Methanococcus maripaludis orMethanocaldococcus jannaschii.
 7. The method of claim 6, wherein theSepRS is comprises an amino acid sequence at least 85% identical to SEQID NO:43 or
 46. 8. (canceled)
 9. The method of claim 1, wherein thenucleic acid encoding a gene with tRNA^(Sep) activity and the nucleicacid encoding a gene with SepRS activity are on one or more vectors. 10.The method of claim 9, wherein the vector is an expression vectorselected from the group consisting of a plasmid, a virus, a nakedpolynucleotide, and a conjugated polynucleotide.
 11. The method of claim9, wherein the vector is expressed in cells selected from the groupconsisting of bacterial cells, archeaebacterial cells, and eukaryoticcells.
 12. The method of claim 9, wherein the vector is expressed in anin vitro transcription/translation system.
 13. The method of claim 12,wherein the vector is transcribed and translated prior to or along withnucleic acids encoding one or more proteins or polypeptides.
 14. Themethod of claim 1, wherein the nucleic acids are expressed in anorganism.
 15. The method of claim 1, wherein the nucleic acids are undercontrol of a promoter selected from the group consisting ofconstitutive, inducible and tissue-specific.
 16. A kit for producing atarget protein containing phosphoserine, comprising a polynucleotideencoding EF-Sep, a polynucleotide encoding tRNA^(Sep), and apolynucleotide encoding SepRS.
 17. The kit of claim 16, wherein the kitfurther comprises phosphoserine.
 18. The kit of claim 16, furthercomprising a host system for expressing a polynucleotide encoding theprotein, the polynucleotide encoding EF-Sep, the polynucleotide encodingtRNA^(Sep), and the polynucleotide encoding SepRS.
 19. A plurality of atarget protein produced according to the method of claim
 1. 20. Theplurality of the target protein of claim 19 in a lysate of host cellsused to produce the target protein.
 21. A method comprising screeningcandidate drugs for activity against a protein, wherein the protein isproduced according to a process comprising the method of claim 1.